Combined Inhibition of mTOR and CDK4/6 Is Required for Optimal Blockade of E2F Function and Long-term Growth Inhibition in Estrogen Receptor-positive Breast Cancer.

The cyclin dependent kinase (CDK)-retinoblastoma (RB)-E2F pathway plays a critical role in the control of cell cycle in estrogen receptor-positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor-resistant cell lines reactivate the CDK-RB-E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908-20. ©2018 AACR.CRUK Cambridge Institute, Cambridge, UK (Jason Carroll, Igor Chernukhin, Rasmus Siersbæk3) Novo Nordisk Fonden (NNF 14136) (Rasmus Siersbæk

BRAF(E600)-associated senescence-like cell cycle arrest of human naevi

Most normal mammalian cells have a finite lifespan(1), thought to constitute a protective mechanism against unlimited proliferation(2-4). This phenomenon, called senescence, is driven by telomere attrition, which triggers the induction of tumour suppressors including p16(INK4a) (ref. 5). In cultured cells, senescence can be elicited prematurely by oncogenes(6); however, whether such oncogene-induced senescence represents a physiological process has long been debated. Human naevi ( moles) are benign tumours of melanocytes that frequently harbour oncogenic mutations ( predominantly V600E, where valine is substituted for glutamic acid) in BRAF(7), a protein kinase and downstream effector of Ras. Nonetheless, naevi typically remain in a growth-arrested state for decades and only rarely progress into malignancy (melanoma)(8-10). This raises the question of whether naevi undergo BRAF(V600E)- induced senescence. Here we show that sustained BRAF(V600E) expression in human melanocytes induces cell cycle arrest, which is accompanied by the induction of both p16(INK4a) and senescence- associated acidic beta-galactosidase (SA-beta-Gal) activity, a commonly used senescence marker. Validating these results in vivo, congenital naevi are invariably positive for SA-beta-Gal, demonstrating the presence of this classical senescence-associated marker in a largely growth-arrested, neoplastic human lesion. In growth-arrested melanocytes, both in vitro and in situ, we observed a marked mosaic induction of p16(INK4a), suggesting that factors other than p16(INK4a) contribute to protection against BRAF(V600E)- driven proliferation. Naevi do not appear to suffer from telomere attrition, arguing in favour of an active oncogene-driven senescence process, rather than a loss of replicative potential. Thus, both in vitro and in vivo, BRAF(V600E)-expressing melanocytes display classical hallmarks of senescence, suggesting that oncogene-induced senescence represents a genuine protective physiological process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62941/1/nature03890.pd

The essence of senescence

Almost half a century after the first reports describing the limited replicative potential of primary cells in culture, there is now overwhelming evidence for the existence of “cellular senescence” in vivo. It is being recognized as a critical feature of mammalian cells to suppress tumorigenesis, acting alongside cell death programs. Here, we review the various features of cellular senescence and discuss their contribution to tumor suppression. Additionally, we highlight the power and limitations of the biomarkers currently used to identify senescent cells in vitro and in vivo

Oncogene-induced senescence relayed by an interleukin-dependent inflammatory network.

Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPbeta cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16(INK4A)-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer

PTPN14 is a negative regulator of YAP.

<p>A) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transfected with two independent siRNAs against YAP and analysed in a Resazurin and luciferase assay 72 h after transfection. mRNA levels of YAP were determined by QRTPCR. Luciferase results are normalized based on the Resazurin results. All results are the average of 5 experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test for each siRNA; * p<0.0001. B) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transduced with lentivirus encoding for dox-inducible PTPN14 expression. After pharmacological selection, luciferase expression was measured for both cell lines in the presence and absence of dox (upper panel). A Resazurin assay was carried out in parallel for each sample and used to normalize the luciferase readings. Results are shown as the average of three independent experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.0001. RLU: relative luciferase units. PTPN14 expression achieved by dox and YAP levels in each sample was analyzed by Western blot (lower panel). Tubulin serves as loading control C) 293A cells were transduced with lentivirus encoding dox-inducible PTPN14 expression. YAP localization in control and PTPN14-expressing cell lines was analysed 72 hours post dox induction by IF at low density using confocal miroscopy. D) 293A cells were transduced with lentivirus encoding two independent dox-inducible shRNAs targeting PTPN14. The nuclear/cytoplasmic ratio of YAP was quantified at high density 72 hours post dox induction using a Cellomics automated imager with a conventional microscope, and expressed relative to the control shGFP (left upper panel). Results are shown as the average of 3 independent experiments ± STDEV. For each experiment, the average ratio was calculated from three wells per sample (10 images per well). Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.001, ** p<0.05. Western blot analysis of PTPN14 levels, indicating the efficiency of each shRNA (left lower panel). Tubulin serves as a loading control. Confocal microscopy images of cells 72 hours post dox induction (right panel).</p

YAP-PTPN14 binding is mediated through the WW domain-PPxY motif interaction.

<p>A) 293A cells were transfected with WT GST-PTPN14 and the indicated V5-YAP constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various YAP mutants with WT PTPN14. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. B–C) 293A cells were transfected with WT V5-YAP and the indicated GST-PTPN14 constructs (WT and mutants). A V5 IP was carried out and blotted for GST to study the interaction of the various PTPN14 mutants with WT YAP. Input lanes were loaded with 10% of the amount of lysate used for each IP and used to compare the expression levels of each construct. All lanes in (C) are from a single blot and exposure. D) An SF268 cell line stably expressing the YAP-responsive MCAT_Luc reporter was transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. Luciferase expression of each cell line was analysed 72 hours post dox induction (left panel). A Resazurin assay was carried out in parallel for each sample and used to normalize the luciferase readings. PTPN14 expression levels achieved for each construct were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ΔPTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as loading control. Luciferase results are shown as the average of at least 3 independent experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. E) The mRNA levels of the indicated YAP target genes were assessed in cells from (D) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.001; **p<0.05, F) 293A cells were transduced with lentivirus encoding for the indicated dox-inducible PTPN14 expression. The nuclear/cytoplasmic YAP ratio was quantified at low density after 72 hours of dox induction using a Cellomics automated imager with a conventional microscope, and expressed relative to control (left panel). Results are shown as the average of three experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05. For each experiment, the average ratio was calculated from three wells per sample (10 images per well). PTPN14 expression levels were analysed by Western blot (right panel; arrows indicate the WT PTPN14 protein and the truncated ΔPTP PTPN14 which migrates faster; all lanes from a single blot and exposure). Tubulin serves as a loading control G) Confocal microscopy images of 293A cells from (F) generated 72 hours post dox induction. H) The mRNA levels of the indicated YAP target genes were assessed in cells from (F) 72 hours post dox induction. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05.</p

PTPN14 is a YAP-binding protein.

<p>A) QPCR-based YAP copy number analysis. MCF-10A is used as a control cell line with no YAP amplification. B) Clonogenic survival assay of SF268 cells transduced with lentivirus encoding control or two independent dox-inducible shRNAs (upper panel). QRTPCR analysis of YAP mRNA levels (lower panel). C) Coomassie-stained gel used for MS analysis of YAP-binding proteins. YAP is indicated by the black arrow, PTPN14 by the black arrowhead and the AMOT family members by an asterisk. D) IP of endogenous YAP from SF268 cell lysates. All lanes are from a single blot and exposure. Asterisk indicates the position of the IgG heavy chains.</p

Down-regulation of PTPN14 enhances the oncogenic phenotype of YAP.

<p>A) MCF-10A cells were transduced with lentivirus encoding for YAP expression and after pharmacological selection transduced with lentivirus encoding for dox-inducible shRNAs targeting either PTPN14 or GFP (control) (two independent shRNAs targeting PTPN14). All six cell lines were subjected to an anoikis assay in low-attachment plates in the presence of dox for 48 hours. Results are shown as the average of three experiments ± STDEV. Statistical analysis was carried out with a 2-tailed paired t-test; * p<0.05, ** p = 0.05. B) Western blot analysis of cells from (A) 48 hours post dox induction. Tubulin serves as a loading control. C) Soft agar assay quantification of cells from (A). Results were obtained two weeks post dox induction and are the average of three experiments ± STDEV. D) Images of wells from (C) at the time of quantification. Scale: 1 mm.</p