Novel role of PAF1 in attenuating radiosensitivity in cervical cancer by inhibiting IER5 transcription

Abstract

Background: Radiosensitivity is limited in cervical cancer (CC) patients due to acquired radiation resistance. In our previous studies, we found that immediate-early response 5 (IER5) is upregulated in CC cells upon radiation exposure and decreases cell survival by promoting apoptosis. The details on the transcriptional regulation of radiation-induced IER5 expression are unknown. Studies in recent years have suggested that Pol II-associated factor 1 (PAF1) is a pivotal transcription factor for certain genes "induced" during tumor progression. In this study, we investigated the role of PAF1 in regulating IER5 expression during CC radiotherapy. Methods: PAF1 expression in CC cells was measured by western blotting, immunohistochemistry, and qRT-PCR, and the localization of PAF1 and IER5 was determined by immunofluorescence. The effect of PAF1 and IER5 knockdown by siRNA in Siha and Hela cells was studied by western blotting, qRT-PCR, CCK-8 assay, and flow cytometry. The physical interaction of PAF1 with the IER5 promoter and enhancers was confirmed using chromatin immunoprecipitation and qPCR with or without enhancers knockout by CRISPR/Cas9. Results: We confirmed that PAF1 was highly expressed in CC cells and that relatively low expression of IER5 was observed in cells with highly expressed PAF1 in the nucleus. PAF1 knockdown in Siha and Hela cells was associated with increased expression of IER5, reduced cell viability and higher apoptosis rate in response to radiation exposure, while simultaneous PAF1 and IER5 knockdown had little effect on the proportion of apoptotic cells. We also found that PAF1 hindered the transcription of IER5 by promoting Pol II pausing at the promoter-proximal region, which was primarily due to the binding of PAF1 at the enhancers. Conclusions: PAF1 reduces CC radiosensitivity by inhibiting IER5 transcription, at least in part by regulating its enhancers. PAF1 might be a potential therapeutic target for overcoming radiation resistance in CC patients.

FOXC2/ADAM12‐dependent radiosensitivity of head and neck squamous cell carcinoma cells

“…A previous study showed that Pol II-associated factor 1 was upregulated in human cervical tumors and reduced the radiosensitivity of cervical cancer cells by inhibiting the transcription of immediate-early response 5. 26 Forkhead box protein M1 (FOXM1) and kinesin family member 20A (KIF20A) were highly expressed in irradiated human lung cancer cell lines and FOXM1 reduced the radiosensitivity of lung cancer cells partially by upregulating KIF20A. 27 These findings show the promise of protein regulators as therapeutic targets in the radiotherapies for human cancers.…”

“…FOXC2 and ADAM12 were both upregulated in response to radiation, and knockdown of FOXC2 inhibited the proliferative ability of irradiated HNSCC cells. A previous study showed that Pol II‐associated factor 1 was upregulated in human cervical tumors and reduced the radiosensitivity of cervical cancer cells by inhibiting the transcription of immediate‐early response 5 26 . Forkhead box protein M1 (FOXM1) and kinesin family member 20A (KIF20A) were highly expressed in irradiated human lung cancer cell lines and FOXM1 reduced the radiosensitivity of lung cancer cells partially by upregulating KIF20A 27 .…”

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

“…The function of PAF1C in hindering pause release was initially found by forward genetic screens in zebrafish, showing that PAF1C antagonizes the release of erythroid genes (32). This was later confirmed by reverse genetic screens in human cells using highly paused genes as readouts (12) and supported by studies demonstrating inhibitory roles of PAF1C in transcription especially pausing in varying contexts (21,22,28,(39)(40)(41)(42)(43). However, the role of PAF1C in promoting pause release has also been reported in several studies under different biological settings (17,22,23,44).…”

R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

“…In S. pombe, previous studies have found that high dNTP levels can lead to increased DNA damage sensitivity and slow progression through S-phase (Fleck et al, 2013;Pai and Kearsey, 2017). A recent study has suggested a role for PAF1 in attenuating radiosensitivity by inhibiting immediate-early response 5 (IER5) transcription, which is involved in cell cycle regulation (Ding et al, 2019;Zheng et al, 2020).…”