| Abstract |
Activation of a telomere maintenance mechanism is a vital step in the
development of most cancers and provides a target for the selective
killing of cancer cells. Cancers can use either telomerase or Alternative
Lengthening of Telomeres (ALT) to maintain their telomeres and inhibition
of either telomere maintenance mechanism can cause cancer cells to undergo
senescence or apoptosis. Although telomerase inhibitors are undergoing
clinical trials, on commencing this study very little was known about the
role of ALT in cancer, what proteins were involved in its mechanism and
regulation and how it could be targeted clinically. The primary aim of
this thesis was to develop an assay for ALT suitable for examining
archived tumour specimens and to begin using it to examine the prevalence
and clinical significance of ALT in cancer. This assay and gene expression
analysis was also used to identify genes that are involved in or
associated with the activation of the ALT mechanism, to contribute towards
the overall goal of an ALT cancer therapy. The ALT mechanism involves
recombination mediated replication and ALT cells have a marked increase in
a range of recombinational events specifically at their telomeres.
Presumably, as a consequence of this the telomere lengths of ALT cells are
very heterogeneous and on average long. This can be detected by terminal
restriction fragment (TRF) Southern analysis, which has been used
previously as the definitive test for ALT activity. However, TRF analysis
requires intact genomic DNA and is unsuitable for tumour specimens which
are commonly archived by paraffin embedding. Another hallmark of ALT is
ALT-associated PML bodies (APBs) which are the subset of PML bodies that
contain telomeric DNA. Work done in this study to consolidate APBs as a
hallmark of ALT, combined with published data, showed 29/31 ALT[+], 3/31
telomerase[+] and 0/10 mortal cell lines/strains are APB[+]. The three
APB[+]/telomerase[+] cell lines identified here had an order of magnitude
lower frequency of APB[+] nuclei than the ALT[+] cell lines. APBs may be
functionally linked to the ALT mechanism and contain the recombination
proteins that are thought to be involved in the ALT mechanism. This study,
in collaboration with Dr W-Q Jiang, strengthened this functional link by
demonstrating that loss of ALT activity (as determined by TRF analysis)
coincided with the disruption of APBs. The detection of APBs was developed
into a robust assay for ALT in archived tumour specimens using a technique
of combined immunofluorescence and telomere fluorescence in situ
hybridisation. It was demonstrated that the APB assay concurred exactly
with the standard assay for ALT (TRF analysis) in 60 tumours for which TRF
analysis gave unequivocal results. The APB assay may be a more appropriate
technique in the case of tumour specimen heterogeneity, which may explain
why the APB assay was able to give definitive results when TRF analysis
was equivocal. We demonstrated that intratumoral heterogeneity for ALT
does exist and this could explain why about 3% of tumours in this study
were APB[+] but with more than a ten-fold reduction in the frequency of
APB[+] nuclei. This study also made the novel discovery of single stranded
C-rich telomeric DNA inside APBs which potentially could be used to make
the APB assay more suitable for routine pathology laboratory use. The APB
assay was used to show that ALT is a significant concern for oncology. ALT
was utilised in approximately one quarter of glioblastoma multiforme
(GBM), one third of soft tissue sarcomas (STS) including three quarters of
malignant fibrous histiocytomas (MFH), half of osteosarcomas and one tenth
of non-small cell lung carcinomas (NSCLC). Furthermore, the patients with
these ALT[+] tumours had poor survival; median survivals were 2 years for
ALT[+] GBM, 4 years for ALT[+] STS including 3.5 years for ALT[+] MFH and
5 years for ALT[+] osteosarcoma. ALT[+] STS and osteosarcomas were also
just as aggressive as their ALT[-] counterparts in terms of grade and
patient outcome. ALT status was not found to be associated with response
to chemotherapy in osteosarcomas or survival in STS. ALT was however, less
prevalent in metastatic STS. The APB assay was a prognostic indicator for
GBM and was correlated with three fold increased median survival in GBM
(although this survival was still poor). ALT was more common in lower
grade astrocytomas (88% ALT[+]) than GBM (24% ALT[+]) and ALT[+] GBM had
an identical median age at diagnosis to that reported for secondary GBM.
It is discussed that these data indicate that ALT was indirectly
associated with secondary GBM and is possibly an early event in its
progression from lower grade astrocytoma. This is relevant because
secondary GBM have distinct genetic alterations that may facilitate
activation of the ALT mechanism. Putative repressors of ALT could explain
why this study found that ALT varied among the different STS subtypes. ALT
was common in MFH (77%), leiomyosarcoma (62%) and liposarcoma (33%) but
rare in rhabdomyosarcoma (6%) and synovial sarcoma (9%). ALT was not found
in colorectal carcinoma (0/31) or thyroid papillary carcinoma (0/17) which
have a high prevalence of telomerase activity and a reduced need for a
telomere maintenance mechanism (low cell turnover), respectively. A yeast
model of ALT predicts that one of the five human RecQ helicases may be
required for ALT. Using the APB assay to test for the presence of ALT in
tumours from patients with known mutations in either WRN or RECQL4 it was
demonstrated that neither of these RecQ helicases is essential for ALT.
Although p53 and mismatch repair (MMR) proteins have been suggested to be
possible repressors of ALT, there was no apparent increase in the
frequency of ALT in tumours from patients with a germline mutation in p53
codon 273 or in colorectal carcinomas that had microsatellite instability
and thus MMR deficiency. Also contrary to being a repressor of ALT but
consistent with its ability to interact with a protein involved in the ALT
mechanism, the MMR protein MLH1, was demonstrated to be present in the
APBs of an ALT[+] cell line. To further test for genes that may be
involved in the ALT mechanism or associated with its activation, RNA
microarray was used to compare the gene expression of 12 ALT[+] with 12
matched telomerase[+] cell lines; 240 genes were identified that were
significantly differentially expressed (p<0.005) between the ALT[+] and
telomerase[+] cell lines. Only DRG2 and SFNX4 were significantly
differentially expressed after adjusting for the estimated false positive
rate. Overall, DRG2, MGMT and SATB1 were identified as most likely to be
relevant to the ALT[+] tumours and Western analysis indicated that DRG2
and MGMT levels were down-regulated after activation of ALT and up-
regulated after activation of telomerase, whereas SATB1 protein levels
appeared to be up-regulated after immortalisation but to a higher degree
with activation of ALT compared to telomerase. Since lack of MGMT is known
to be a determinant of temozolomide sensitivity in GBM, the possibility
that ALT and the APB assay could be used to predict temozolomide
sensitivity is discussed. The microarray data was consistent with MGMT
expression being suppressed by EGF (p < 0.05), indicating that caution may
be needed with combining EGFR inhibitors with temozolomide in ALT cancers.
One ALT[+] cell line which did not express MGMT had TTAA sequence in its
telomeres. This could possibly have resulted from mutations due to lack of
MGMT expression and a possible role for MGMT in the ALT mechanism is
discussed. Further analysis of the microarray data identified two groups
of co-regulated genes (p < 5x10-5): CEBPA, TACC2, SFXN4, HNRPK and MGMT,
and SIGIRR, LEF1, NSBP1 and SATB1. Two thirds of differentially expressed
genes were down-regulated in ALT. Chromosomes 10 and 15 had a bias towards
genes with lower expression in ALT while chromosomes 1, 4, 14 and X had a
bias towards genes with higher expression levels in ALT. This work has
developed a robust assay for ALT in tumour specimens which was then used
to show the significance of ALT in sarcomas, astrocytomas and NSCLC. It
has also identified genes that could possibly be molecular targets for the
treatment of ALT[+] cancers.
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