Implicações de mutações na Claspina na Manutenção da Homeostasia Celular, Oncogénese e Resposta ao Tratamento

Cancer remains one of the leading causes of death worldwide. Genomic instability is a hallmark of most cancers and constitutes a source of genetic variability that can foster malignant transformation or provide adaptive and survival abilities to cancer cells. To maintain genomic integrity and prevent DNA damage, cells rely on checkpoints that function as barriers that halt cell cycle progression whenever damage is detected. The cell cycle remains blocked until damage is repaired or the affected cell is eliminated, preventing the perpetuation of errors.Claspin is a multifunctional protein critically needed for checkpoint activation, acting as a vital scaffold protein that mediates the phosphorylation of the effector kinase Chk1 by ATR. Claspin also has a role in DNA repair following DNA damage checkpoint activation, functionally connecting the components of the checkpoint response and DNA repair pathways. If the damage is not repairable, Claspin degradation is required for apoptosis induction. Claspin is a key component in the maintenance of genomic integrity. Claspin participates actively in the fork protection complex, acting as a sensor and promoting replication stability by coupling DNA unwinding to strand polymerization, and may also indirectly regulate replication initiation at firing origins. Claspin is therefore a multifunctional protein that participates in essential processes involved in maintenance of cellular homeostasis. Such processes are frequently altered in cancer and evidence shows that Claspin may have a significant role in carcinogenesis. A body of evidence suggests that Claspin inactivation is an essential event during carcinogenesis, indicating that Claspin may function as a tumor suppressor. However, other studies have detected high levels of Claspin expression in tumors, which were associated with worse prognosis and parameters of cancer progression, indicating that Claspin may have a more complex role in cancer than previously anticipated. CLSPN genetic alterations can modulate or alter Claspin availability and/or function. However, little is known regarding the impact of genetic alterations on its function, particularly in the context of cancer. In this study, we aimed to study the functional impact of genetic alterations on Claspin’s function and its possible association with cancer. For that, CLSPN germline alterations were firstly characterized in samples of patients presenting sporadic or familial breast cancer, or glioma, as well as in six cancer cell lines. Eight variants were identified (c.-68C>T, c.17G>A, c.1574A>G, c.2230T>C, c.2028+16G>A, c.3595-3597del, and c.3839C>T). Of these, CLSPN c.2028+16G>A was significantly associated with familial breast cancer and glioma; c.2230T>C (p.Ser744Pro) was exclusively detected in breast cancer and glioma patients, but not in healthy controls; and CLSPN c.1574A>G (p.Asn525Ser) was significantly associated with breast cancer. Bioinformatic tools were used to predict a potential functional impact of these variants. Polyphen-2 classified CLSPN c.1574A>G as a genetic alteration with probably damaging effects, and Alamut and HExoSplice softwares predicted that this variant could have a significant impact on CLSPN mRNA splicing. Therefore, this variant was further studied using a minigene assay to assess the impact of CLSPN c.1574A>G on exon splicing. We observed that this variant caused partial skipping of exon 8. Additionally, Claspin p.Asn525Ser variant was associated with decreased Claspin expression and decreased Chk1 activation. Although bioinformatic analysis did not classify the CLSPN promoter genetic alteration, c.-68C>T, as a variant with functional impact, we have decided to study this variant, as promoter mutations have the potential to modulate the transcriptional activity of genes. Indeed, we have observed that this variant increased the transcriptional activity of CLSPN’s promoter in HeLa and HEK cells, in a luciferase assay. However, no significant differences were detected in U2OS cells.In conclusion, CLSPN variant CLSPN c.1574A>G seems to modulate Claspin’s availability and function by altering CLSPN mRNA processing. This might lead to decreased Claspin expression and, consequently, Chk1 activation. In turn, the c.-68C>T promoter variant appears to represent a gain of function genetic alteration, suggesting a complex role for Claspin in carcinogenesis, which is further supported by the literature. The role of Claspin in cancer seems to be dependent on the cellular context, the mutational landscape of tumors, and the stage of oncogenesis. In addition, Claspin seems to have a role in the tumors’ response to chemo- and radiation therapy. Currently available data suggest that Claspin-targeted therapies hold great potential for radio- and chemo-sensitization, especially in situations in which Claspin is implicated in the mechanisms of therapeutic resistance.