Abstract

Background

Results

Conclusions

Methods and Approach

According to CD31 & Twist1 staining, we did not find any significant difference in vesicular density or EMT features.

According to Ki67 staining, Ptch1+/- visible BCCs metastasized at the slowest rate in comparison to other genotyped groups of mice.

Acknowledgements: Thank you to our wonderful lab and all of the brilliant members of it. A thank you to the NIH for funding Epstein lab research over the previous years.

References: (1). “Basal cell carcinomas: attack of the hedgehog”, Ervin H. Epstein, Nature Review, 2008.

Basal Cell Carcinoma (BCC) is the most prevalent cancer amongst people of European ancestry; a non-melanocytic skin cancer that originates in basal cells of the skin epidermis. Aberrant activation of hedgehog (HH) signaling plays a pivotal role in BCC carcinogenesis. Besides mutations in genes encoding HH pathway components, mutations in the p53 gene frequently are found in human BCCs. Consistently, previous studies in Epstein Lab have established that loss of p53 dramatically accelerates BCC formation in ionizing radiation treated Ptch1+/- mice. Additionally, our recent data showed that loss of Arf, another tumor suppressor gene, functioning upstream of p53 and mediating oncogene-induced stress pathway, also achieved tumor promotion effects via both p53-dependent and -independent mechanisms. Here in this particular study, we further characterized the visible BCCs developed in these mice with loss of p53, loss of Arf, or loss of both p53 and Arf compared to those developed in Ptch1+/- mice (i.e. p53+/+ Arf+/+) using immunohistochemistry.

Figure 1a.(1)

Normal Hedgehog Signaling. The extracellular ligand Hedgehog binds to the transmembrane receptor PTCH1. This binding deactivates the inhibition of smoothened (SMO), which results in intracellular signaling towards SUFU and Gli . If Sonic Hedgehog ligand fails to bind to PTCH1, then PTCH1 sends inhibitory signals towards SMO and stops the intracellular signaling

Basal Cell Carcinomas (BCCs) remain the most common skin cancer amongst our human population. Regions of BCC development occur in UV-exposed areas of the dermal layer, typically aggregating towards the head and neck regions. Standard treatment of these non-melanocytic tumors involves surgical removal of the affected dermal lesion. There is a low reoccurence rate of BCCs on post-operative patients, provided that the entirety of the dermal lesion is intially removed.

Objective: To characterize the visual BCCs biopsed from four different genotyped groups of Ptch1+/- mice.

1). Wildtype= Ptch1+/-

2). PF= Ptch1+/- K14CreER2 p53 fl/fl

3). ARF= Ptch1+/- K14CreER2 Arf fl/fl

4). DD= Ptch1+/- K14CreER2 p53 fl/fl Arf fl/fl

Figure 2. Molecular Genotyping. LoxP sites flank the tumor supressor genes (e.g. p53, Arf). Mouse is injected with Tamoxifen through intraperonital method at 6 weeks of age. Upon injection, Tam binds to Cre-ER2 receptor, which activates the excising function of the Lox P sites. The Lox P sites flank the targeted gene intended to be excised or alters the functionality of the promoter.

Results

Results

As expected, all visible BCCs displayed upregulation of Gli1 expression due to abnormal HH pathway activation.

Future Directions

LoxPLoxP p53*

Tumor Suppressor Function of Arf and p53 in Basal Cell Carcinoma (BCC) Development in Ptch1+/- MiceRachelle Vazquez1, Grace Wang1, Flora Ting1, John Dolorito1, Vaishnavi Sitarama1, Ervin Epstein1

1Children’s Hospital & Research Center Oakland, Center for Cancer, Oakland, CA, USA

Cre-ER2

Figure 1b.(1) Abberant Hedgehog Signaling. The extracellular ligand Sonic Hedgehog binds to the transmembrane receptor PTCH 1, which disinhibits smoothened intracellular signaling. If Sonic Hedgehog ligand fails to bind to PTCH 1, then PTCH 1 sends inhibitory signals towards smoothened and stops the intracellular signaling cascade.

✂ ✂

Promoter

(1)

(1)

Figure 4b. CD31 Vessel Staining. Vesicular staining illuminated by green flurorescence.K14 Keratinocyte staining illuminated by magenta pigmentation.

Causes of BCC proliferation include abnormal Hedgehog pathway activation and loss of functional tumor supressor genes such as p53 and arf. Aberrant activation of hedgehog signaling can result from Ptch1+/- mice that have a one copy deletion of Ptch1 gene, resulting in upregulation of Hedgehog signaling. This increase in HH pathway signaling can also lead to expression of SMO (potentially activating mutations) and overexpression of Gli1 or Gli2 in keratinocytes. The Oncogenic Induced Stress Pathway (OIS) is mediated by tumor supressor gene p53, which in turn functions to mediate Arf in p53-dependent pathway mechanisms.

Ptch1+/- PF ARF

Figure 4a. CD31 Vessel Staining. CD31 immunohistological staining quantifies neovascularization (formation of new blood vessels from endothelium) in metastasized tumor tissue.

Figure 4c. CD31 Vessel Staining. Vesicular staining illminated by green flurorescence. Purple pigmentation reflects nuclei staining amongst tumor nests in biopsed frozen sections of tissue.

Recombinant LoxP

Quantification of CD31 vesicular staining results in comparison to Ptch1+/- control group.

Alternative EMT staining using Snail, Slug, qPCR, etc.

CD31 Neovascularization

Figure 3. Immunohistochemistry Methods. Different staining protocols were implemented in order to determine the level of activation of specialized biological function.

Tamoxifen

Figure 5a. Ki67 Cell Proliferation Staining. PF & ARF mice display the highest density of positive Ki67 staining within visible BCC tissue.

Figure 5b. Ki67 Cell Proliferation Staining. Ptch1+/- mice display the lowest density of positive Ki67 staining within visible BCC tissue.

Figure 6a. Twist1 EMT Staining. Strong positive staining amongst stroma of cellular tumor nests.

Methods: TAM at 6 weeks, IR at 8 weeks. Skin biopsies taken at 4 months. Observation for visible BCCs.

a) Immunofluorescence (IF): CD31 (1:200) frozen sections

b) Immunohistochemistry (IHC): Ki67 (1:200) FFPE Ptch vs. PF, ARF, DD and Twist1 (1:200) FFPE

c) qPCR Hedgehog Signaling (HH) of Gli1

Figure 6b. Twist1 EMT Staining. PF displays strong positive staining in selective stromal amongst n tumor nests.

Twist1

Ki67

EMT Signaling

Cell Proliferation

Figure 2. Quantification of Gli1 Levels. The amount of Gli1 transcription factors correlates to a relief of disinhibited SMO signaling through the bound PTCH1 transmembrane receptor. Over-expression of Gli1 in keratinocytes has been previously linked to BCC proliferations. There is no statistical difference between all of the mouse gropus.

Figure 6c. Twist1 EMT Staining. ARF displays the strongest positive staining of Twist1 in the cellular stroma.