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Robinson et al. J Cancer Metastasis Treat 2019;5:39 I http://dx.doi.org/10.20517/2394-4722.2019.15 Page 3 of 9

CSCs, like embryonic and tissue stem cells, possess replicative immortality , a process achieved in part
[42]
by activating telomere maintenance mechanisms (TMMs) [43,44] . As outlined below, TMMs function within
a network that unites cellular immortalization with processes, including EMT, that drive the development
and outgrowth of metastatic cells. Telomeres, therefore, serve as essential mediators of CSC maintenance
and consequent metastatic evolution. In addition, the results detailed below implicate telomere homeostasis
as an attractive target for novel therapeutics to treat metastatic breast cancer.

TELOMERES AND TELOMERE DYNAMICS IN CSCS AND METASTATIC CELLS
Telomeres are nucleoprotein complexes located at the ends of linear chromosomes that safeguard against
chromosomal instability and the loss of genetic information during cell division . In humans, the DNA
[45]
component of telomeres is composed of tandem (TTAGGG) repeats with a 3' single-stranded overhang that
n
invades telomeric duplex DNA to form a protective loop . These DNA regions are coated with proteins that
[46]
collectively constitute the shelterin complex. Shelterin proteins serve to shield telomeres from illicit activation
of DNA damage responses (DDRs); they also maintain genome integrity and recruit factors responsible for
regulating telomere length [47,48] . In somatic (i.e., non-immortalized) cells, telomeres shorten during iterative
rounds of cell division. To combat this event, stem cells and cancer cells maintain their telomeres using one
of two TMMs: telomerase or alternative lengthening of telomeres (ALT). Telomerase is a reverse transcriptase
enzyme composed of an RNA moiety (TERC, also known as TR) that provides a template for telomeric
DNA synthesis and a protein moiety (TERT) that facilitates telomerase recruitment and carries out its
polymerase activity . In contrast, ALT relies upon homology-directed, recombination-dependent synthesis
[49]
of nascent telomeric DNA . ALT requires transient deprotection of telomeres coupled to activation of a
[50]
DDR that is accompanied by telomere extension in a manner similar to break-induced DNA synthesis [51,52] .
DDR activation occurs in response to alterations in telomeric and subtelomeric chromatin structure that
are brought about by loss of the chromatin remodelers ATRX and DAXX [53,54] . Notably, evidence of each of
these mechanisms has been found in breast cancer and can be correlated with specific histologic subtypes or
disease stages [55,56] . These findings support the idea that TMM identity may impact breast cancer progression,
including the onset of metastasis.

While TMM acquisition has been identified as a feature of both stem and non-stem cancer cells, these
processes play an essential role in preferentially sustaining the CSC population . By virtue of their
[42]
replicative immortality, CSCs function as progenitors that exist over a sufficient timescale for evolution
to take place. Remarkably, telomere shortening appears to be a primary driving force underlying tumor
evolution. Telomere shortening precedes TMM activation , which allows for the formation of critically
[57]
short telomeres that cannot be adequately capped by shelterin. Cells interpret these short telomeres as free
DNA ends, which are temporarily repaired by chromosome end-to-end fusions that ultimately induce
breakage-fusion-bridge (BFB) cycles [58,59] . BFB cycling leads to complex genomic rearrangements including
deletions, non-reciprocal translocations, and formation of dicentric or circular chromosomes . Telomere
[60]
catastrophe may also yield chromosomal instability that is resolved via chromothripsis or other forms of
chromoanagenesis, an event termed telomere crisis [61,62] . Breast cancer-initiating cells can harbor both short
telomeres and telomerase activity , consistent with the model that telomere shortening instigates genomic
[41]
instability and CSC evolution while telomere elongation maintains emergent CSC subpopulations [Figure 1]
Evidence identifying ALT in breast CSCs has not yet been found. However, ALT has a stem cell origin ,
[63]
while ALT activity has been observed in non-breast CSCs [44,63,64] . Future studies examining TMMs in breast
CSCs and their connection to genome architecture and tumor heterogeneity will be of great value.

Telomere maintenance proteins have been heavily implicated in many of the central signaling pathways
in metastasis [Figure 1]. For instance, TERT is capable of regulating Wnt target genes by forming a
[65]
transcriptional co-activation complex with β-catenin . In addition, TERT directly regulates NF-κB-
[66]
dependent gene expression by binding to the NF-κB p65 subunit at the promoters of target genes [67,68] .