Laboratory of Tumor Progression

Adam Glick, Ph.D.

I was recently recruited to The Pennsylvania State University from the National Cancer Institute, where I was a Principle Investigator in the Laboratory of Cellular Carcinogenesis and Tumor Promotion. My research at the NCI and here in the Center for Molecular Toxicology and Carcinogenesis focuses on the molecular mechanisms and signaling pathways that regulate progression of squamous tumors from a benign to malignant phenotype. Nonmelanoma skin cancer is increasing in the United States at alarming rates due to higher UV light exposure. In addition immunosuppression of organ transplant recipients leads to highly aggressive recurrent cutaneous squamous cell carcinoma in these patients. Thus understanding the signaling pathways controlling squamous tumor progression is critical to develop new therapeutic strategies.

The major biological model that I use is multistage skin carcinogenesis in the mouse. In this model cancers are generated in the skin of mice by topical application of carcinogens and repeated exposure to chemicals that cause outgrowth of benign tumors, some of which eventually progress to malignant squamous cell carcinomas. The mouse epidermis serves as a useful model for human cancers since most solid tumors form in epithelial tissues, are frequently the result of some type of environmental or physical carcinogen exposure and progress though multiple stages. Due to the superficial nature of the mouse skin tumors, the different stages can be easily quantitated and manipulated. In addition many transgenic and knockout mice can be utilized to understand the role of a specific gene in cancer development. My lab is specifically focused on signaling pathways that are critical for tumor progression.

Stages in the Pathogenesis of Squamous Cancer

Normal skin Papilloma Squamous Cell Spindle Cell

Carcinoma Carcinoma

The Transforming Growth Factor-b (TGFb1) signaling pathway is a critical regulator of tumor development in humans and the mouse multistage skin carcinogenesis model TGFb1 is a growth factor secreted by normal and neoplastic cells that has multiple biological effects on many different cell types. It has a unique property of being a negative regulator of cell growth. Many human cancers are no longer responsive to TGFb1 due to specific mutations in the signaling pathway. However, many human cancers produce high levels of TGFb1 which affect the immune system and the normal cells surrounding the tumor in a way that favors tumor growth. An additional level of complexity arises from the multiple signaling pathways that interact either in a positive or negative way with the TGFb1 pathway to alter transcriptional responses to this growth factor, and ultimately influence the behaviour of the neoplastic cell. Understanding the molecular basis for the multiple functions of TGFb1 during tumor development is a major focus of my laboratory.

Schematic of the TGFb1 Signaling Pathway. TGFb is secreted as part of a latent complex that requires processing for bioactive TGFb1 to bind to the type II receptor or TbRII. Once bound to ligand this constitutively active serine-threonine kinase recruits and phosphorylates the type I receptor, also known as TbRI or ALK5. Phosphorylation of TbR1 activates its serine-threonine kinase activity towards the Smad proteins, transcription factors that shuttle from the cytoplasm to the nucleus in response to phosphorylation by the receptor. Smad2 and 3 phosphorylated by the TGFb receptor and Activin receptor, while Smads 1, 5 and 8 are phosphorylated by BMP receptors. All receptor activated Smads complex with Smad4 and this complex translocates to the nucleus where transcriptional regulation occurs at SBE sites, and at non-Smad recognition sites through interaction with other transcription factors. Counteracting these signals are the inhibitory Smads, Smad6 and Smad7. These Smads bind to the type I receptors and block phosphorylation of the R-Smads. Smad7 can block multiple pathways, while Smad6 is restricted to the BMP pathway. Since these inhibitory Smads are induced by activation of the pathway, it is thought that they act to generate a negative feedback loop.

To study the role of TGFb1 in the pathogenesis of squamous cancer our laboratory uses genetic means to disrupt the TGFb1 signaling pathway such as the TGFb1 -/- or Smad3 -/- mice or overexpression of Smad7 via a retrovirus. We use in vivo and in vitro bioassays to determine how these changes alter tumor progression in the context of keratinocytes initiated with a ras oncogene.

We have recently developed a conditional expression model that allows regulated expression of bioactive TGFb1 as well as other genes in the mouse epidermis. In this model the doxycycline regulated transactivator rTA is expressed in the epidermis with the keratin 5 promoter, while a constitutively active porcine TGFb1 cDNA linked to the tetO binding site for the rTA is the regulated target gene. In K5/rTA x tetOTGFb1 bitransgenic mice, doxycycline is required for transactivation of TGFb1. For the K5/tTA line, doxycyline suppresses transactivation while removal allows induction of the target gene.

Induction of TGFb1 in the adult causes inflammation and a reversible alopecia. This conditional expression system affords precise control of the timing of transgene induction thereby allowing more sophisticated studies of target gene effects on cancer development than were possible with conventional transgenics. Using this system we find that induction of TGFb1 during gestation produces a lethal phenotype while induction in the adult causes inflammation and a reversible alopecia. We have used these mice to induce TGFb1 expression in benign and malignant squamous tumors and have used a genomics approach to identify gene expression responses to TGFb that are different between the two tumor types. These studies show a significant difference in the immune response when TGFb1 is overexpressed in papillomas and SCC. Current projects involve understanding how tumor produced TGFb1 differentially effects the immune system, and how the interaction of TGFb1 and other signaling pathways control the altered gene expression profiles.

One of the most important signaling pathways that interact with TGFb is that of oncogenic ras. In the skin carcinogeneis model, the c-Ha ras gene is frequently mutated by chemical carcinogens such as DMBA. Interactions between the TGFb1 and oncogenic ras signaling pathways are important for both the tumor suppressive and pro-oncogenic effects of TGFb1 in cancer development. Current projects in the lab involve a genomic analysis of the effects of oncogenic ras on the transcriptional response to TGFb1 in primary keratinocytes. We find oncogenic ras causes gene and promoter specific inhibition of TGFb1 signaling and are investigating downstream effectors of this inhibitory effect. We are also using the conditional expression mouse model to co-express the ras oncogene and TGFb1 in the normal epidermis to test how these signaling pathways interact in vivo