Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRAS(G12V) dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRAS(G12V) sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-beta pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRAS(G12V) exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53

Atypical E2Fs either Counteract or Cooperate with RB during Tumorigenesis Depending on Tissue Context

Simple Summary In virtually all human malignancies, the CDK-RB-E2F pathway is dysregulated resulting in the activation of the E2F transcriptional network. Rb and atypical E2Fs are the most important negative regulators of E2F-dependent transcription during tumorigenesis. However, it is unknown whether they cooporate or act independently in tumor development. Here we show that combined loss of RB and atypical E2Fs in mice enhances tumorigenesis in the liver, while in the pituitary gland, we observe inhibition of tumorigenesis. These findings suggest that the interaction between RB and atypical E2Fs in controlling tumorigenesis occurs in a tissue cell-type specific manner. E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency

CDC6:A novel canine tumour biomarker detected in circulating extracellular vesicles

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours

Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRAS G12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRAS G12V sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-β pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRAS G12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage

Graphical Abstract Highlights d Individual cycling cancer cells display enhanced E2F target gene expression d E2F7/8 deletion or E2F3 overexpression overrides cell-cycle exit after DNA damage d Elevated levels of the E2F target Emi1 prevent DNA-damage-induced cell-cycle exit d The cell-cycle exit after DNA damage is transient and leads to endoreplicatio

Oncogenic RAS sensitizes cells to drug-induced replication stress via transcriptional silencing of P53

Cancer cells often experience high basal levels of DNA replication stress (RS), for example due to hyperactivation of oncoproteins like MYC or RAS. Therefore, cancer cells are considered to be sensitive to drugs that exacerbate the level of RS or block the intra S-phase checkpoint. Consequently, RS-inducing drugs including ATR and CHK1 inhibitors are used or evaluated as anti-cancer therapies. However, drug resistance and lack of biomarkers predicting therapeutic efficacy limit efficient use. This raises the question what determines sensitivity of individual cancer cells to RS. Here, we report that oncogenic RAS does not only enhance the sensitivity to ATR/CHK1 inhibitors by directly causing RS. Instead, we observed that HRAS G12V dampens the activation of the P53-dependent transcriptional response to drug-induced RS, which in turn confers sensitivity to RS. We demonstrate that inducible expression of HRAS G12V sensitized cells to ATR and CHK1 inhibitors. Using RNA-sequencing of FACS-sorted cells we discovered that P53 signaling is the sole transcriptional response to RS. However, oncogenic RAS attenuates the transcription of P53 and TGF-β pathway components which consequently dampens P53 target gene expression. Accordingly, live cell imaging showed that HRAS G12V exacerbates RS in S/G2-phase, which could be rescued by stabilization of P53. Thus, our results demonstrate that transcriptional control of P53 target genes is the prime determinant in the response to ATR/CHK1 inhibitors and show that hyperactivation of the MAPK pathway impedes this response. Our findings suggest that the level of oncogenic MAPK signaling could predict sensitivity to intra-S-phase checkpoint inhibition in cancers with intact P53

Atypical E2Fs either Counteract or Cooperate with RB during Tumorigenesis Depending on Tissue Context

E2F-transcription factors activate many genes involved in cell cycle progression, DNA repair, and apoptosis. Hence, E2F-dependent transcription must be tightly regulated to prevent tumorigenesis, and therefore metazoan cells possess multiple E2F regulation mechanisms. The best-known is the Retinoblastoma protein (RB), which is mutated in many cancers. Atypical E2Fs (E2F7 and -8) can repress E2F-target gene expression independently of RB and are rarely mutated in cancer. Therefore, they may act as emergency brakes in RB-mutated cells to suppress tumor growth. Currently, it is unknown if and how RB and atypical E2Fs functionally interact in vivo. Here, we demonstrate that mice with liver-specific combinatorial deletion of Rb and E2f7/8 have reduced life-spans compared to E2f7/8 or Rb deletion alone. This was associated with increased proliferation and enhanced malignant progression of liver tumors. Hence, atypical repressor E2Fs and RB cooperatively act as tumor suppressors in hepatocytes. In contrast, loss of either E2f7 or E2f8 largely prevented the formation of pituitary tumors in Rb+/- mice. To test whether atypical E2Fs can also function as oncogenes independent of RB loss, we induced long-term overexpression of E2f7 or E2f8 in mice. E2F7 and -8 overexpression increased the incidence of tumors in the lungs, but not in other tissues. Collectively, these data show that atypical E2Fs can promote but also inhibit tumorigenesis depending on tissue type and RB status. We propose that the complex interactions between atypical E2Fs and RB on maintenance of genetic stability underlie this context-dependency

Atypical E2f functions are critical for pancreas polyploidization

The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2ftranscription factors, E2f7and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7or E2f8alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age

Feedback regulation between atypical E2Fs and APC/C-Cdh1 coordinates cell cycle progression

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C-Cdc20. Importantly, atypical E2Fs can activate APC/C-Cdh1 by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C-Cdh1, which ensures balanced expression of cell cycle genes and normal cell cycle progression