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CD34+ cell chaacterization in K5-IKKbeta skin.

Study of SCCs in Ink4a/ArfKO background.

Primer sequences.

Immune cell populations in wt and K5-IKKbeta skin.

Legends to Supplementary Figures and supplementary Material and Methods.

Expression of IKKbeta, K13 and Phospho-AKT in papillomas and carcinomas of p53EKO and p53EKO/K5-IKKbeta mice.

Tumor multiplicity, histopathological charecterization and size distribution in Ink4a/Arfwt/wt and Ink4a/Arfwt/wt/K5-IKKbeta mice.

Supplementary Table 4: Spreadsheet of transcriptomic data

Includes Supplementary Figures 1-13; Supplementary Tables 1, 2, 3, 7 and 8; and Supplementary References

Purpose:Bladder cancer is a clinical and social problem due to its high incidence and recurrence rates. It frequently appears in elderly patients showing other medical comorbidities that hamper the use of standard chemotherapy. We evaluated the activity of CDK4/6 inhibitor as a new therapy for patients unfit for cisplatin (CDDP).Experimental Design:Bladder cancer cell lines were tested for in vitro sensitivity to CDK4/6 inhibition. A novel metastatic bladder cancer mouse model was developed and used to test its in vivo activity.Results:Cell lines tested were sensitive to CDK4/6 inhibition, independent on RB1 gene status. Transcriptome analyses and knockdown experiments revealed a major role for FOXM1 in this response. CDK4/6 inhibition resulted in reduced FOXM1 phosphorylation in vitro and in vivo and showed synergy with CDDP, allowing a significant tumor regression. FOXM1 exerted important oncogenic roles in bladder cancer.Conclusions:CDK4/6 inhibitors, alone or in combination, are a novel therapeutic strategy for patients with advanced bladder cancer previously classified as unfit for current treatment options.

Supplementary Table 5: Spreadsheet of transcriptomic data

Supplementary Figures 1-4 from Spontaneous Squamous Cell Carcinoma Induced by the Somatic Inactivation of Retinoblastoma and Trp53 Tumor Suppressors

Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia with poor prognosis and survival that frequently displays Akt overactivation. Here we show that mice displaying constitutive Akt activity (myrAkt) in combination with Trp53 loss in stratified epithelia develop oral cavity tumors that phenocopy human HNSCC. The myrAkt mice develop oral lesions, making it a possible model of human oral dysplasia. The malignant conversion of these lesions, which is hampered due to the induction of premature senescence, is achieved by the subsequent ablation of Trp53 gene in the same cells in vivo. Importantly, mouse oral tumors can be followed by in vivo imaging, show metastatic spreading to regional lymph nodes, and display activation of nuclear factor-κB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-β type II receptor expression, thus resembling human counterparts. In addition, malignant conversion is associated with increased number of putative tumor stem cells. These data identify activation of Akt and p53 loss as a major mechanism of oral tumorigenesis in vivo and suggest that blocking these signaling pathways could have therapeutic implications for the management of HNSCC. [Cancer Res 2009;69(3):1099–108]

Supplementary Figure 1 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Supplementary Legends 1-2 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Supplementary Figure 2 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage. [Cancer Res 2007;67(22):10879–88]

Supplementary Table 1 from Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organ-transplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53- and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer. [Cancer Res 2008;68(3):683–92]

Supplementary Figures 1-10 from Akt Activation Synergizes with Trp53 Loss in Oral Epithelium to Produce a Novel Mouse Model for Head and Neck Squamous Cell Carcinoma

Supplementary Data - Gene List from Spontaneous Squamous Cell Carcinoma Induced by the Somatic Inactivation of Retinoblastoma and Trp53 Tumor Suppressors

IKKβ (encoded by IKBKB) is a protein kinase that regulates the activity of numerous proteins important in several signaling pathways, such as the NF-κB pathway. IKKβ exerts a protumorigenic role in several animal models of lung, hepatic, intestinal, and oral cancer. In addition, genomic and proteomic studies of human tumors also indicate that IKBKB gene is amplified or overexpressed in multiple tumor types. Here, the relevance of IKKβ in skin cancer was determined by performing carcinogenesis studies in animal models overexpressing IKKβ in the basal skin layer. IKKβ overexpression resulted in a striking resistance to skin cancer development and an increased expression of several tumor suppressor proteins, such as p53, p16, and p19. Mechanistically, this skin tumor–protective role of IKKβ is independent of p53, but dependent on the activity of the Ink4a/Arf locus. Interestingly, in the absence of p16 and p19, IKKβ-increased expression favors the appearance of cutaneous spindle cell–like squamous cell carcinomas, which are highly aggressive tumors. These results reveal that IKKβ activity prevents skin tumor development, and shed light on the complex nature of IKKβ effects on cancer progression, as IKKβ can both promote and prevent carcinogenesis depending on the cell type or molecular context. Implications: The ability of IKKβ to promote or prevent carcinogenesis suggests the need for further evaluation when targeting this protein. Mol Cancer Res; 15(9); 1255–64. ©2017 AACR.

Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia with poor prognosis and survival that frequently displays Akt overactivation. Here we show that mice displaying constitutive Akt activity (myrAkt) in combination with Trp53 loss in stratified epithelia develop oral cavity tumors that phenocopy human HNSCC. The myrAkt mice develop oral lesions, making it a possible model of human oral dysplasia. The malignant conversion of these lesions, which is hampered due to the induction of premature senescence, is achieved by the subsequent ablation of Trp53 gene in the same cells in vivo. Importantly, mouse oral tumors can be followed by in vivo imaging, show metastatic spreading to regional lymph nodes, and display activation of nuclear factor-κB and signal transducer and activator of transcription-3 pathways and decreased transforming growth factor-β type II receptor expression, thus resembling human counterparts. In addition, malignant conversion is associated with increased number of putative tumor stem cells. These data identify activation of Akt and p53 loss as a major mechanism of oral tumorigenesis in vivo and suggest that blocking these signaling pathways could have therapeutic implications for the management of HNSCC. [Cancer Res 2009;69(3):1099–108]