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Project

Targeting radiotherapy resistance in Head and Neck Cancers: the potential biomarker role of TIPRL1 and CIP2A, two cellular inhibitors of Protein Phosphatase 2A

Being one of the main post-translational modifications, reversible protein phosphorylation is of major importance in signal transduction regulation. Over the years, the kinases phosphorylating many substrates have been extensively studied, while the protein phosphatases have been lagging behind. The PP2A-like phosphatases PP2A, PP4 and PP6, all have been shown to play a role in the regulation of tumorigenesis and the DNA damage response (DDR). Since many cancers including head and neck squamous cell carcinoma (HNSCC) are treated with DNA damaging agents, those phosphatases and their regulators could serve as attractive therapeutic targets improving treatment response.

In this thesis, we focused on two cellular inhibitors of PP2A, CIP2A and TIPRL1, of which the latter is also known to inhibit PP4 and PP6. First, we showed TIPRL1 expression is increased in HNSCC tumor tissues compared to normal tissue. Patients with TIPRL1-high expressing tumors showed lower locoregional control and survival upon radiotherapy. This observation was further validated in an HPV-negative HNSCC cell line (SQD9), which showed radiotherapy sensitization after TIPRL1 depletion. This sensitization correlated with faster cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Moreover, more γH2AX foci remained in the TIPRL indel cell lines 24h after irradiation, although this phenotype was not rescued by reintroduction of TIPRL1. Next, we identified a role for TIPRL1 in ATM signaling, as we found that TIPRL1 is phosphorylated by ATM kinase at Ser265 upon radiotherapy or upon treatment with cisplatin or a PARP inhibitor. This phosphorylation was required for TIPRL1-mediated radiotherapy resistance. Furthermore, two new TIPRL1 interaction partners, DNA-PKcs and RAD51, were identified using mass spectrometry analysis and validated by immunoblotting. Both interactions were induced by radiotherapy and remained unaffected by TIPRL1 phosphorylation. In contrast, the nucleosomal histones were also identified as new TIPRL1 interaction partners upon irradiation, but their interaction was adversely affected by TIPRL1 phosphorylation. As expected, the PP2A-like phosphatases belonged to TIPRL1’s interactome as well, but their interaction was not influenced by radiotherapy nor by TIPRL1 phosphorylation. Interestingly, we found that PP6 could bind TIPRL1 both in the presence and the absence of PPIs, while PP2A and PP4 could only interact with TIPRL1 in the absence of PPIs, suggesting a different binding mode and, potentially, a distinct mechanism of inhibition/regulation, which should be further investigated.

We also found increased radiosensitization upon CIP2A depletion from SQD9 cells. CIP2A indel cells showed lower cell survival and plating efficiency without treatment, which was even more pronounced when treating the cells with 4 and 6 Gy irradiation. In contrast to TIPRL1 depletion, CIP2A depleted cells showed less γH2AX foci shortly (1 to 3h) after 2 Gy irradiation and lower checkpoint kinase phosphorylation, corresponding with a PP2A activating effect. As reactivation of PP2A is an attractive new target, we aimed to validate whether bortezomib and erlotinib decreased cell viability through CIP2A downregulation; however, both showed similar efficiency in CIP2A-depleted cells, suggesting that their effects occurred independently of CIP2A downregulation.

Finally, we showed that three alleged small molecule activators of PP2A, ATUX792, SMAP2 and iHAP, also decreased cell viability independently of CIP2A and TIPRL1 expression, except for ATUX792, which showed a little less efficiency in the absence of TIPRL1. As a next step, a combination treatment of ATUX792 and radiotherapy could be tested in HNSCC cells to improve radiotherapy response.

Overall, our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in HNSCC RT resistance through modulation of DNA damage checkpoint activation and repair. Also, CIP2A mediates RT resistance in HNSCC via a role in the DNA damage response. Our research will help to guide better, personalized treatments for HPV-negative HNSCC patients, and by extension, for other cancer patients treated with DNA damaging therapies.

Date:1 Jan 2018 →  22 Nov 2023
Keywords:Head and neck squamous cell carcinoma, Protein phosphatase 2A, Disease models
Disciplines:Biochemistry and metabolism, Systems biology, Medical biochemistry and metabolism
Project type:PhD project