Decoding the mechanisms of cancer and stem cell immortality
- Author(s): Chiba, Kunitoshi
- Advisor(s): Hockemeyer, Dirk
- et al.
Telomeres are the repetitive sequences at the ends of linear chromosomes. The
key functions of telomeres are to protect the cells from losing genomic information and to
prevent chromosome ends from being repaired by the double strand break repair
machinery. To counteract loss of telomeric DNA, cells can express a reverse
transcriptase, telomerase, that synthesizes telomeric repeats de novo. In humans,
telomerase activity is mostly restricted to germ and stem cells, so the telomeres of most
somatic cells progressively shorten with each cell division. Once telomeres become
critically short, they are recognized as sites of DNA damage and cells cease to proliferate.
By this mechanism, telomere shortening functions as a tumor suppression mechanism.
TERT, the protein component of telomerase, becomes silenced once stem cells
differentiate. However, in 90% of cancer cells, TERT is transcriptionally re-activated.
Thus, telomerase regulation is crucial for our understanding of telomere length regulation
in stem cell maintenance and tumorigenesis. To understand how telomerase acts on
telomeres, I attempted to endogenously tag telomerase. To do this I inserted epitope tags
at the endogenous TERT locus in hESCs using genome editing. However, I found that all
the tested tags cause defects in telomere maintenance, which was previously not
appreciated in experiments using exogenous overexpression.
Recently, point mutations in the TERT promoter were identified as the most
frequent non-coding mutations in cancer. To elucidate the role of TERT promoter
mutations (TPMs) in tumorigenesis, I genetically engineered these TPMs into human
embryonic stem cells (hESCs) using genome editing. Using the resulting isogenic hESC
lines, I demonstrated that TPMs lead to a failure of TERT silencing upon differentiation
from stem into somatic cells. To understand role of TPMs in tumorigenesis, I monitored
long-term telomere maintenance and proliferation in human fibroblasts engineered to
carry TPMs. I found that TPMs immortalize cells but do not prevent telomere shortening
and telomere fusions. In vitro, around the time when telomere fusions occurred, TERT
expression was gradually increased. Thus, TPMs are required, but not sufficient, for
cancer cell immortality and contribute to tumorigenesis in two steps. First, TPMs expand
proliferation capacity of a cell by elongating only the shortest telomeres but do not
prevent overall telomere shortening. In the second step, TPMs fuel tumorigenesis by not
fully suppressing genomic instability. In order for cells to immortalize they need to
upregulate TERT during this second step.