Telomere dysfunction is one of the hallmarks of cancer and is related to shelterin dysfunction. TRF1 is a protein that binds directly to double-stranded telomeric DNA, interacting directly or indirectly with other shelterin components, telomerase, DNA repair machinery, and other proteins responsible for telomere maintenance . The loss of TERF1 is related to genomic instability, a characteristic related to PC development and progression [5, 6]. It is interesting to note that TERF1 is in the 8q.21 region and amplifications in that region of the chromosome have already been associated with a worse prognosis for PC . In the present study, we tried to understand the TERF1 role in PC.
First, we observed a great increase in the number of amplifications in TERF1 on CRPC. These results may lead us to conclude that TERF1 amplification is an important event in the acquisition of the lethal phenotype in PC, where cancer evolves to metastasis resistant to treatments. Since normally, the patients with PC only die when the disease progress to CRPC, we next check whether alterations on TERF1 can impact the overall survival of these patients .
The survival analysis suggests that amplifications in TERF1 are associated with cancer aggressiveness only on the primary PC. The probable reason for the large proportion of alterations in the CRPC is due because it is exactly these more aggressive tumors that evolve to the lethal phenotype, but the amplification itself does not change the prognosis for these patients.
Then, we demonstrate a downregulation of TERF1 in both TCGA and clinical cohorts of localized PC. This phenomenon is expected since the downregulation of TERF1 can leads to increase in genetic instability and may favor the cellular immortalization, remarkable features at the onset of prostate carcinogenesis, with similar observations observed in breast cancer [5, 6].
On the contrary, the upregulation of TERF1 may be related with poor prognosis on primary PC, being associated with poor overall and disease-free survival, lymph node invasion, and AR upregulation.
Besides that, we showed a correlation between TERF1 and AR only on cancer tissue. This is relevant because studies showed that androgen axis modulates telomere length in normal cells, which suggest that AR control telomeres . Specifically on prostate, it is postulated that AR repress the expression of telomerase in normal cells, but reactivates the enzyme in cancer cells, which favors cancer initiation and progression . In addition, AR interacts directly with shelterin through TIN2 (which binds directly to TRF1) and AR antagonist promotes telomere dysfunction only in AR positive PC cell lines [5, 16]. Our result reinforce that AR modulates telomere dynamics during prostate cancer progression, and, maybe TERF1 plays a crucial role in this process.
In the clinical cohort, we corroborate that miR-155 overexpression can causes the depletion of TERF1. MiR-155 is a well-known oncomiR and has been shown to be overexpressed in many cancers, including PC . Despite the extensive literature about this microRNA, it is most commonly associated with the regulation of genes related to inflammation, the immune system and hematopoiesis . In this study, we showed that miR-155 is upregulated in primary PC samples that do not show any detectable expression of TERF1.
Despite the importance of microRNAs in several biological processes, the relationship between them and telomeres is not well studied. For example, in addition to TERF1 and miR-155, only three others microRNAs have already been validated by regulating other shelterin genes (miR-490 and miR-23a for TERF2 and miR-185 for POT1) [21,22,23]. In addition, there are studies that show that telomerase is regulated by several microRNAs, which indicates that the interaction between telomeres and microRNAs is a vast field that should be more explored in future studies.
Considering metastatic PC, our results support the possibility that TERF1 expression is important for telomere maintenance and cancer cell immortalization in advanced cancer stages, by protecting the cell of telomeric DNA damage and promoting cancer cell stemness.
The induction of telomere uncapping by TERF1 genetic depletion has been shown to effectively block the initiation and progression of aggressive tumors in lung and glioblastoma mouse models, and some studies have recently proposed the induction of TERF1 depletion as a possible molecular treatment in glioblastoma multiforme [24, 25].
On the contrary, during tumor progression to advanced stages, telomere shortening, chromosome instability, and increased tumor aggressiveness had been related to progressive downregulation of TERF1. The promotion of cancer aggressiveness by TERF1 downregulation already been reported in metastatic PC models . Our results using metastatic cell lines reinforce this theory. In the three most important PC cell lines, we were able to show that there is a progressive downregulation of TERF1 from a castration-sensitive cell line to the castration-resistant cell lines. In breast cancer, TERF1 downregulation is related to higher genomic instability and an increase in radioresistance [6, 26]. In PC, there has only been one study showing that overexpression of TERF1 is related to unfavorable prognostic factors . Here, we also showed that low TERF1 expression was related to a better prognostic on localized PC.
We advocate that TERF1 depletion and telomeric dysfunction are important for the first steps of carcinogenesis (localized PC) and are supplanted by other dysfunctions during tumor progression.
After that, probably to avoid genetic crisis, some cancer cells recover TERF1 expression, probably by an AR-dependent mechanism, and progress to an aggressive phenotype with a certain level of genomic stability (such as seen in LNCaP cells and advanced localized tumors). Here, it is important to remember that, even though genetic instability is a hallmark of the cancer cell, very high levels of genomic instability can be harmful to cancer progression for several reasons (such as the increase of neoantigens).
Suggesting a mechanism, we postulate that overexpression of miR-155 suppresses TERF1 expression in the carcinogenesis process. This would favor genetic instability and cell immortalization promoting cancer initiation. After that, cells with AR upregulation increase the expression of TERF1, suppressing the excess of DNA damage at telomeres. Considering that TERF1 cannot inhibit telomerase activity (a tumor suppressor effect) without PINX1 (and that this protein is downregulated in advanced stages of the PC), this phenomenon can promote the evolution to a more aggressive stage of the cancer . It is interesting to note that a recent study reports that TERF2 modulates PC progression by regulating telomere DNA damage pathways, which reinforce the involvement of shelterin in disease progression .
Amplifications of TERF1 were enriched in CRPC datasets, but these alterations were not too relevant in this stage of the cancer. Another important characteristic of CRPC is that AR amplification/reactivation is very common. In DU145 and PC3 (CRPC cell lines), we showed a downregulation of TERF1 which is coherent. In these two cell lines, the downregulation of TERF1 is probably associated with the resume of genetic instability, present in both cells, and the fact that they are AR-negative.