Mechanisms of decreased expression of transforming growth factor-beta receptor type I at late stages of HPV16-mediated transformation.
Transforming growth factor-beta (TGF-beta) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-beta resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-beta during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-beta sensitivity in HKc/DR is attributed to decreased expression of TGF-beta receptor type I (TGF-beta RI), while the levels of TGF-beta receptor type II (TGF-beta RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-beta RI expression in HKc/DR. Using TGF-beta RI and TGF-beta RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-beta RI and TGF-beta RII by about 50%. However, promoter activity of TGF-beta RI is decreased to a greater extent than TGF-beta RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-beta RI expression in HKc/DR was found not to be linked to mutations within the TGF-beta RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-beta RI promoter found no changes between HKc/HPV16 and HKc/DR in binding of the transcription factors Sp1 or Sp3 to the probes. Also, Western blots determined that protein levels of Sp1 and Sp3 remain relatively unchanged between HKc/HPV16 and HKc/DR. Overall, these results demonstrate that mutations in or hypermethylation of the TGF-beta RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-beta RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-beta receptor promoter activity.
Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA.
Melissa K
MK
Hypes
Lucia
L
Pirisi
Kim E
KE
Creek
D001665:Binding Sites; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics; D050725:Papillomavirus E7 Proteins; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000032:analysis; D051705:Sp3 Transcription Factor / Q000032:analysis
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D012097:Repressor Proteins; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C059731:oncogene protein E7, Human papillomavirus type 16; D051705:Sp3 Transcription Factor; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II