Periodic Reporting for period 1 - PSIPCancer (Role of PSIP1 in prostate cancer development and drug resistance)
Período documentado: 2021-01-05 hasta 2023-01-04
The focus of the project was on understanding the role of PSIP1, a chromatin factor in transcriptional alteration and maintaining genomic integrity during transcription. PSIP1 is known to have role in transcription elongation, splicing and in HIV integration. But its role in DNA damage repair and genomic instability is not known. Hence, in this project, it aimed to understand the role of this PSIP1 in maintaining genomic integrity during transcription.
Understanding the factors that are involved in maintaining genomic integrity is very important and these factors play crucial role in cancer development and treatment response. By modulating such factors, we will be able to develop novel treatment modalities or improving the outcome of existing treatment approaches in cancer. Therefore, this project was aimed at understanding the role of PSIP1 in transcriptional alteration and in maintaining genomic integrity in the sites of transcription.
The overall objectives of the project included, studying the altered transcription mediated by PSIP1 through enhancer activation, studying the role of PSIP1 in maintain genome integrity during transcription and the role of PSIP1 in drug resistance in cancer cells. The depletion of PSIP1 resulted in increased R-loops, increased DNA damage leading to reduced synthesis of nascent transcripts at the gene bodies, indicating the novel and important role of PSIP1 in R-loop homeostasis and transcription. This project has also showed the role of PSIP1 in repairing of the DNA damage at the transcribing regions by facilitating homologous repair pathway thereby emphasising its role in transcription. In light of these findings, this project has further demonstrated that the PSIP1 deficient cancers can be targeted better using PARP1 inhibitors and drugs that cause transcription coupled damage.
Understanding the factors that are involved in maintaining genomic integrity is very important and these factors play crucial role in cancer development and treatment response. By modulating such factors, we will be able to develop novel treatment modalities or improving the outcome of existing treatment approaches in cancer. Therefore, this project was aimed at understanding the role of PSIP1 in transcriptional alteration and in maintaining genomic integrity in the sites of transcription.
The overall objectives of the project included, studying the altered transcription mediated by PSIP1 through enhancer activation, studying the role of PSIP1 in maintain genome integrity during transcription and the role of PSIP1 in drug resistance in cancer cells. The depletion of PSIP1 resulted in increased R-loops, increased DNA damage leading to reduced synthesis of nascent transcripts at the gene bodies, indicating the novel and important role of PSIP1 in R-loop homeostasis and transcription. This project has also showed the role of PSIP1 in repairing of the DNA damage at the transcribing regions by facilitating homologous repair pathway thereby emphasising its role in transcription. In light of these findings, this project has further demonstrated that the PSIP1 deficient cancers can be targeted better using PARP1 inhibitors and drugs that cause transcription coupled damage.
Using an RNA-seq analysis the transcriptional change mediated by PSIP1 in PC-3 and LNCaP prostate cancer cells as compared to normal cells was studied to understand the role of PSIP1 in mediating the cancer specific transcriptional alteration. CUT&Tag-seq for H3K27ac, H3K4me1, H4K16Ac, γH2AX and androgen receptor in control and PSIP1 KD prostate normal and cancer cells was also performed, to know the aberrant enhancer activation. Since this analysis indicated a role for PSIP1 in DNA repair, a proteomics study from immunoprecipitation of endogenous PSIP1 was performed. It identified several proteins involved in transcription, RNA processing, DNA repair and R-loop homeostasis to be interacting with PSIP1. Therefore, the role of PSIP1 in R-loop homeostasis was investigated. Immunoprecipitation and proximity ligation assay (PLA) confirmed the interaction of PSIP1 with the R-loop complex. In normal prostate epithelial cells (RWPE-1) depleted with PSIP1 (PSIP1-KD), resulted in significant increase in R-loop levels as compared to the control as seen by slot blot, immunofluorescence and CUT&Tag-seq. This PSIP1 depletion mediated accumulation of R-loops lead to accumulation of DNA damage in those sites as seen by γ-H2AX western blotting and CUT&Tag. Notably, PSIP1 is enriched at γ-H2AX peaks specific to PSIP1-KD, confirming that γ-H2AX accumulation is due to the direct consequence of PSIP1 depletion. Furthermore, PSIP1 KD cells showed increased number of PLA foci between γ-H2AX and R-loops implying the co-localisation of γ-H2AX and R-loop in PSIP1-KD. This data further confirmed that R-loops that accumulate upon PSIP1 depletion are responsible for increased DNA damage in PSIP1 KD. RNase H1 overexpression and inhibition of RNAPII transcription by triptolide rescued the elevated level of γ-H2AX demonstrating that the elevated DNA damage in the absence of PSIP1 is due to R-loop accumulation and is RNAPII transcription-dependent. Further analysis on genomic context revealed that PSIP1 binding is enriched around gene promoters, and PSIP1 depletion led to an increase in R-loop levels and DNA damage at those promoter sites and gene bodies. Transient transcriptome sequencing (TT-seq) revealed a stark reduction in the nascent transcript levels at R-loop peaks detected explicitly in the PSIP1-KD. Further results indicated that this accumulation of R-loops lead to the collision between transcription and replication machinery leading to the DNA damage. Further, PSIP1 was involved in repair of these DNA damage by facilitating homologous repair pathway. Hence, the sensitivity of cancer cells with low PSIP1 to PARP1 inhibitors and clastogens that cause transcription-coupled DNA damage. Results showed that the prostate cancer cells with lower levels of PSIP1, to more sensitive to PARP1 inhibitor – Olaparib compared to prostate normal cells. Depletion of PSIP1 led to hypersensitivity of prostate cancer cells to illudin-S –a drug that cause transcription coupled DNA damage.
The results of these projects were regularly discussed and disseminated in Lab meetings, joint Lab meetings, QMUL Epigenetics-Hub meetings, Blizard Institute centre seminars and at the Gordon International Research conference on Chromatin Structure and Function.
The results of these projects have been published two open access publications.
The results of these projects were regularly discussed and disseminated in Lab meetings, joint Lab meetings, QMUL Epigenetics-Hub meetings, Blizard Institute centre seminars and at the Gordon International Research conference on Chromatin Structure and Function.
The results of these projects have been published two open access publications.
Prostate cancer is the most commonly diagnosed cancer among men in Europe and is a leading cause of cancer related deaths. Due to the resistance development against the existing drugs, devising novel therapeutic targets and treatment modalities is very important. This project has showed novel insights into the transcriptional alteration and mechanism through which PSIP1 maintains genome integrity at the site of transcription. In light of these insights, the project has also shown that the cancers with low levels of PSIP1 are sensitive to Olaparib and drugs that cause transcription coupled DNA damage demonstrating the clinical implications of these findings. Based on these synthetic lethal interactions, novel cancer treatment modalities can be devised or the efficacy of existing treatment outcomes can be improved and thereby future health of citizens and quality of life can be improved.