PHARMACOLOGICAL INHIBITION OF THE TGF-BETA TYPE I RECEPTOR REVEALS A DUAL ROLE OF TGF-BETA IN CARCINOGENESIS
Open Access
- Author:
- Markell, Lauren Mordasky
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 05, 2010
- Committee Members:
- Adam Bleier Glick, Dissertation Advisor/Co-Advisor
Adam Bleier Glick, Committee Chair/Co-Chair
John Patrick Vanden Heuvel, Committee Member
Gary H Perdew, Committee Member
Zhi Chun Lai, Committee Member
Jeffrey Maurice Peters, Committee Member - Keywords:
- skin
skin carcinogenesis
TGF-beta
chemically-induced carcinogenesis - Abstract:
- Transforming growth factor-beta 1 (TGF-beta 1) is a member of a large family of regulatory molecules that play both positive and negative roles in epithelial cancer. Pharmacological inhibitors of the Transforming Growth Factor-beta (TGF-beta) type I receptor (ALK5) have shown promise in blocking growth of xenotransplanted cancer cell lines but the effect on a multistage cancer model is not known. In the present studies, the role of the ALK5 inhibitor SB431542 (SB) was investigated in a two-stage skin chemical carcinogenesis assay in order to determine the effect on tumor formation and progression. In this model, nearly all tumors have activating mutations in codon 61 of the HRas gene. We show that topical SB significantly reduced the total number, incidence and size of papillomas compared to 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion alone, and this was linked to increased epidermal apoptosis, decreased proliferation and decreased cutaneous inflammation during promotion. In contrast, the frequency of conversion to squamous cell carcinoma (SCC) was two-fold higher in papillomas treated with SB. While there was no difference in tumor cell proliferation in early premalignant lesions, those that formed after SB treatment exhibited reduced squamous differentiation and an altered inflammatory microenvironment similar to SCC. In an inducible epidermal HRAS transgenic model, treatment with SB enhanced proliferation and cutaneous inflammation in skin, but decreased expression of keratin 1 and increased expression of simple epithelial keratin 18, markers of premalignant progression. In agreement with increased frequency of progression in the multistage model, SB treatment resulted in increased tumor formation with a more malignant phenotype following long-term HRAS induction. To further characterize the altered squamous differentiation associated with reduced tumor formation and increased malignant progression by ALK5 inhibition, an HRAS oncogene-induced model of preneoplastic keratinocytes was utilized. SB significantly enhanced HRAS-induced cornification which correlated with the increased expression of terminal differentiation genes transglutaminase 1 (TGM1) and 3 (TGM3) and small proline-rich protein 1A (SPR1A) and 2H (SPR2H) which cross-link structural proteins that make up the cornified envelope. There was a similar increase in cornified layers and TGM and SPR gene expression following SB treatment of mice expressing inducible epidermal HRAS. Alternatively, treatment of HRAS-expressing keratinocytes with TGF-beta 1 or overexpression of TGF-beta 1 and HRAS in an inducible epidermal transgenic model resulted in reduced expression of TGM1 and TGM3 and cornification in vitro. Papilloma (SP1) and squamous cell carcinoma (PAM2.12) cell lines were less responsive to TGF-beta 1 suppression of markers and marker induction by SB. Interestingly, a subpopulation of HRAS-expressing keratinocytes were resistant to induction of terminal differentiation by ALK5 inhibition. These cells were also resistant to senescence and rapidly immortalized, suggesting that they represent a progressed phenotype. However, these cells were unable to grow in elevated calcium or produce tumors after grafting nude mice indicating that they were not fully malignant. Thus, selective responsiveness to differentiation may represent a mechanism by which blocked TGF-beta signaling can inhibit the outgrowth of preneoplastic lesions but may cause a more progressed phenotype in a separate keratinocyte population. Collectively, these data support the concept that a large number of initiated keratinocytes are dependent on ALK5 signaling for enhanced tumor outgrowth. A second subset can form tumors in part through generation of an altered inflammatory microenvironment and through altered squamous differentiation induced by inhibition of ALK5. This latter group has a higher frequency of malignant conversion. In contrast to the current paradigm for TGF-beta 1 in carcinogenesis, these results demonstrate that cutaneous TGF-beta 1 signaling enables promotion of benign tumors but suppresses premalignant progression through context-dependent regulation of epidermal homeostasis and inflammation.