Telomerase and Telomeres in Endometrial Cancer

Abstract

Telomeres at the termini of human chromosomes are shortened with each round of cell division due to the "end replication problem" as well as oxidative stress. During carcinogenesis, cells acquire or retain mechanisms to maintain telomeres to avoid initiation of cellular senescence or apoptosis and halting cell division by critically short telomeres. The unique reverse transcriptase enzyme complex, telomerase, catalyzes the maintenance of telomeres but most human somatic cells do not have sufficient telomerase activity to prevent telomere shortening. Tissues with high and prolonged replicative potential demonstrate adequate cellular telomerase activity to prevent telomere erosion, and high telomerase activity appears to be a critical feature of most (80-90%) epithelial cancers, including endometrial cancer. Endometrial cancers regress in response to progesterone which is frequently used to treat advanced endometrial cancer. Endometrial telomerase is inhibited by progestogens and deciphering telomere and telomerase biology in endometrial cancer is therefore important, as targeting telomerase (a downstream target of progestogens) in endometrial cancer may provide novel and more effective therapeutic avenues. This review aims to examine the available evidence for the role and importance of telomere and telomerase biology in endometrial cancer.

Keywords: TERRA; TRAP; endometrial cancer; endometrium; hTERC; hTERT; telomerase; telomere.

Figure 1

Schematic illustration of the telomere and main telomerase complex components. The human telomere and telomerase enzyme complex (only one half of the dimeric holoenzyme complex is shown for clarity), adapted from Hapangama et al. (14). From all sheltrin proteins only telomere repeat binding factors 1 (TRF1) and 2 (TRF2) (27) bind directly to the double-stranded telomeric sequence, and protection of telomeres protein-1 (POT1) (28) binds to the single-stranded overhang; hence these are termed as telomere binding proteins and they interact with remaining shelterin proteins TIN2 (binds to TRF1 and TRF2) (29, 30), RAP1 (binds to TRF2) (31) and TPP1 (binds to POT1) (32). The TERC H/ACA region located at the 3′ end binds to dyskerin and the other telomerase associated proteins: NOP10, NHP2, and GAR1 (14). The hTERC at the 3′ end binds also to telomerase Cajal body protein 1 (TCAB1) (33).

Figure 2

Telomere maintenance mechanisms. Cells can maintain their telomeres via either telomerase-dependent pathway or a telomerase-independent ALT pathway. Activated Wnt signaling pathway can maintain telomere length by activating both these maintenance mechanism and by maintaining the level of TRF2 and POT1 sheltrin components that are essential for telomere protection (65). ATM and ATR also have stimulatory effect on telomerase enzyme via triggering its recruitment and enhancing the assembly of this enzyme (66). TERRA binds independently to hTERC and hTERT telomerase subunits with an inhibitory effect on human telomerase enzyme (67) or it acts as a recruiter of telomerase enzyme rather than an inhibitor (68).

Figure 3

The involvement of telomeres and telomerase activity in epithelial cancers. The initial acquisition of tumor promoting mutations is promoted by short dysfunctional telomeres which are subsequently stabilized by high telomerase activity levels that is characteristic for most cancer cells, with the overall result being pre-requisite for unregulated proliferation capacity.

Figure 4

From Valentijn et al. (15). Telomerase inhibitor, Imetelstat affects Telomerase Activity and cell proliferation, but not viability of endometrial epithelial cells. (A) Epithelial cells and (B) stromal cells were maintained in monolayer culture for the indicated times prior to harvesting for TRAP assay. For each time point, n ≥ 4; Patient group 2. (C) Epithelial cells maintained in long-term culture had a phenotype consistent with senescence. Note the enlarged cells and positive blue stain for β-galactosidase in the micrographs (representative of n = 5). (D) Epithelial cells were isolated from an adenocarcinoma of the human endometrium and maintained in culture as a cell line. The cells were treated with the concentrations of Imetelstat indicated for 72 h prior to TRAP. TRAP activity is expressed as a percentage relative to the activity of the mismatch control (mean ± SEM for n = 3 separate experiments). (E) Epithelial cells were maintained in culture for up to 3 days and then treated with 1 μM Imetelstat or mismatch control oligonucleotide for a further 72 h prior to TRAP assay. TRAP activity is expressed as a percentage of the mismatch control (n = 4). T-test, ^*p = 0.02. (F) EEC (n = 5) were directly seeded into 96-well dishes, allowed to attach and treated the next day with Imetelstat or the mismatch control at the concentrations indicated for 72 h. Cell viability was assessed by MTT assay. Note significant loss in cell viability at 100 μM (Mann Whitney test, p = 0.002). (G) Cultures of normal epithelial cells and an adenocarcinoma of the endometrium treated with Imetelstat or mismatch control as before, and immunoblotted for phospho-H3 [phosphohistone H3 (Ser10)]. Histone H3 is only phosphorylated on Ser 10 during mitosis. Shown is a representative blot (top) of normal epithelial cells (n = 5) and the adenocarcinoma (representative of two separate experiments) and densitometric analysis (bottom). T-test, ^**p = 0.009. (H) Stromal cells were grown for 24 h and then treated with 1 μM Imetelstat or mismatch control oligonucleotide for 72 h prior to TRAP assay. Telomerase activity is expressed as a percentage of the mismatch control. T-test, ^***p = 0.0004. This previously published figure in human reproduction (15) is reused with permission.

Figure 5

The role of telomere and telomerase activity in endometrial cancer. Hormonal imbalance (excess of Estrogen or insufficient levels of progesterone) will increase telomerase and elevate telomerase activity was described in all types of endometrial hyperplasia and in endometrial cancer. Dysfunctional telomeres results in genomic instability, the first step in endometrial carcinogenesis. Telomerase dependent pathway is the most widely reported classical telomere length maintenance mechanism but ALT pathway; telomerase independent telomere maintenance was described in some cancer types that lack telomerase activity.

Figure 6

Immunohistochemical staining with an anti-human telomerase antibody in healthy and endometrial tissue samples. Endometrial tissue sections demonstrating hTERT immunostaining in full thickness post-menopausal (PM) section and pipelle biopsy from a patient with endometrial cancer (EC) using a polyclonal rabbit anti-human telomerase antibody (ab27573, Abcam, Cambridge UK), detected with ImmPRESS anti-rabbit polymer and visualization with ImmPACT DAB (Vector Laboratories, Peterborough, UK). Positive nuclear hTERT brownish staining was observed in endometrial normal and cancer glands (red arrow). Magnification ×400, scale bar 10 μm.