MYC Potentiates DNA Damage Response Dependent Cellular Senescence

Abstract

Oncogenesis is the progression of a somatic cell through a finite number of phenotypic stages to becoming a cancer. These stages are defined by genetic and epigenetic lesions that activate oncogenes and disable tumor suppressor functions. Cancer cells can exhibit genomic instability, but the true contribution of genomic instability to cancer development is unknown. Multiple DNA repair pathways ensure genomic integrity and recent evidence suggests that DNA damage response (DDR) pathways serve as an initial barrier to oncogenic stress by inducing cell death or cellular senescence. Cellular senescence is a tumor suppressor mechanism in which precancerous cells enter a permanent state of quiescence. MYC is the prototypical oncogene that functions as a transcription factor to promote unrestrained cellular proliferation and blocks differentiation. Increasing evidence suggests that MYC may also contribute to oncogenesis by inducing genomic instability. Normal primary cells expressing an inducible form of MYC were treated with the DNA replication stress agent hydroxyurea (HU) and/or DDR inhibitors caffeine and UCN-01. Components of the DDR were assayed for phosphorylation status, change in mobility during electrophoresis and protein levels by Western analysis and immunofluorescence. Cell death and cellular senescence were performed using flow cytometry (FACs) and senescence-associated beta-galactosidase (SAB) staining, respectively. MYC augmented DDR signaling following treatment with HU as demonstrated by phospho-western of DDR components. MYC also increased cell death following HU treatment shown by sub-G1 DNA content and annexinV/7-AAD FACs. Similar induction of the DDR was not apparent for other forms of MYC-potentiated cell death not involving DNA damage. MYC potentiating the DDR after treatment with HU was intriguing in light of data suggesting that Ras and BRAF oncogenes cause increased DNA replicational stress resulting in oncogene-induced senescence (OIS) dependent on the DDR. MYC increased numbers of senescent cells using the marker SAB in the presence of HU. Inhibitors of the DDR prevented MYC-dependent increase in cellular senescence, but caused a concomitant increase in cell death. We have demonstrated for the first time that MYC, like the oncogenes Ras and BRAF, induce senescence in normal primary cells. Recent evidence has linked MYC to DNA replication stimulation at the post-translational level. Our data is suggestive that MYC promotes DNA replicational stress thus exciting the DDR signaling pathway and leading to OIS and/or cell death. Taken together our data suggests that OIS may be a general phenomenon resulting from increased DNA replication stress.