ABSTRACT
The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.
Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphotransferases/metabolism , Tumor Suppressor Protein p53/metabolism , cdc25 Phosphatases/metabolism , Cell Movement/drug effects , Cyclin-Dependent Kinase 2/genetics , G1 Phase Cell Cycle Checkpoints/genetics , Gene Expression , Humans , Phosphorylation , Protein Serine-Threonine Kinases , S Phase Cell Cycle Checkpoints/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , cdc25 Phosphatases/geneticsABSTRACT
PURPOSE: Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. EXPERIMENTAL DESIGN: Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. RESULTS: Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. CONCLUSIONS: Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs.
Subject(s)
Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/mortality , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , Human papillomavirus 16/metabolism , Phosphatidylinositol 3-Kinases/genetics , Promoter Regions, Genetic , Adult , Aged , Apoptosis/genetics , Ataxia Telangiectasia Mutated Proteins , Carcinoma, Squamous Cell/virology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation/genetics , Female , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/virology , Human papillomavirus 16/genetics , Humans , Male , Middle Aged , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Radiation Tolerance/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.
