Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence

Abstract

Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc-dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression.oncogene-induced senescence | telomerase | telomere | senescence escape |

Telomere dysfunction promotes transdifferentiation of human fibroblasts into myofibroblasts

“…While normal fibroblasts treated with TGF‐β1 developed dysfunctional telomeres and upregulated α‐SMA expression in a time‐dependent manner, hTERT‐expressing fibroblasts did not develop TIF and showed diminished α‐SMA expression (Figure 3c,d and Supporting Information Figure S3e). Similarly, using human fibroblasts in which dysfunctional telomeres can be repaired using a doxycycline inducible hTERT expression system (Patel et al, 2016), we demonstrate that induction of hTERT expression in TGF‐β1 transdifferentiated myofibroblasts again reduced the levels of α‐SMA expressed in cells (Supporting Information Figure S3f). While the primary function of telomerase is to elongate telomeres and suppress formation of dysfunctional telomeres generated due to telomere shortening and ROS‐induced telomeric replication stress, (Boccardi et al, 2015; Patel et al, 2016), telomerase also possesses noncanonical functions that promote DNA repair and cell survival (Martinez & Blasco, 2011).…”

“…Similarly, using human fibroblasts in which dysfunctional telomeres can be repaired using a doxycycline inducible hTERT expression system (Patel et al, 2016), we demonstrate that induction of hTERT expression in TGF‐β1 transdifferentiated myofibroblasts again reduced the levels of α‐SMA expressed in cells (Supporting Information Figure S3f). While the primary function of telomerase is to elongate telomeres and suppress formation of dysfunctional telomeres generated due to telomere shortening and ROS‐induced telomeric replication stress, (Boccardi et al, 2015; Patel et al, 2016), telomerase also possesses noncanonical functions that promote DNA repair and cell survival (Martinez & Blasco, 2011). To determine whether noncanonical functions of hTERT contribute to the suppression of myofibroblast transdifferentiation, we overexpressed in human BJ fibroblasts DN‐hTERT, a dominant defective mutant of hTERT that inactivates the catalytic activity of hTERT while leaving its noncanonical functions intact (Hahn et al, 1999).…”

“…BJ cells expressing SV40 Large T antigen (Addgene; #1780) and HPV E6 cells were generated by retroviral transductions with pBabe‐Neo‐SV40 Large T antigen plasmid and pBabe‐puro‐HPV‐E6 plasmid, respectively. GM21 cells expressing inducible hTERT were described previously (Patel et al, 2016). To generate fibroblasts in oncogene‐induced senescence, BJ cells were transduced with pBABE‐Puro H‐Ras G12V (Addgene; #1768), selected with puromycin for 48 hr and allowing cells to undergo senescence for 14 days.…”

“…For example, genotoxic stresses that cause DSB formation in telomeric repeats can rapidly activate TDIS, as telomeric breaks resist DNA repair activities (Fumagalli et al, 2012; Hewitt et al, 2012; Suram et al, 2012). Stresses that have been demonstrated to cause TDIS include oncogene expression (Patel, Suram, Mirani, Bischof, & Herbig, 2016; Suram et al, 2012), DNA replication stress (Boccardi et al, 2015; Fumagalli et al, 2012; Hewitt et al, 2012), and reactive oxygen species (ROS) (Boccardi et al, 2015), among others. Whether generated due to progressive telomere erosion or due to DSB formation in telomeric repeats, telomere dysfunction activates a persistent DDR that is mediated by and dependent on p53, a transcription factor that not only trans‐activates DDR genes (Molchadsky, Rivlin, Brosh, Rotter, & Sarig, 2010), but also regulates a wide range of other biological activities including cell differentiation (Rivlin, Koifman, & Rotter, 2015).…”

The Chromatin Landscape of Cellular Senescence

“…However, it was recently reported that some OIS cells acquire chromatin changes that promote the expression of the human telomerase reverse transcriptase gene (hTERT) and, as a consequence, escape senescence [94]. This can have important consequences for tumor formation, as senescent cells share features of the cancer epigenome [35].…”