ASPP1 and ASPP2 link the Ras and p53 signalling pathways

In this thesis, the regulation of ASPP1 and ASPP2 was investigated. ASPP1 and ASPP2 are p53 co-activators that can specifically induce p53 dependent apoptosis but have no effect on p53-dependent cell cycle arrest. Both ASPP1 and ASPP2 contain a Ras-association domain in their amino terminal regions. ASPP1 can bind activated Ras directly via its amino terminal region in vitro, and both endogenous ASPP proteins bind endogenous Ras in vivo after stimulation of cells with serum and growth factors. Oncogenic H-RasV 12 and K-RasV 12 stimulate ASPP1 and ASPP2 pro-apoptotic activity in a p53-dependent manner and can also stimulate ASPP2 co-activation of the p53 family members, p63 and p73. These results suggest that ASPP1 and ASPP2 are novel Ras effector proteins. Ras is upstream of several effector pathways. One of its downstream effector pathways, Raf-MEK-MAPK, can activate ASPP1 and ASPP2. MAPK phosphorylates ASPP2 in vitro, and both ASPP1 and ASPP2 in vivo at serines 746 and 827, respectively. ASPP1 and ASPP2 phosphorylation by MAPK results in an increase in their ability to co-activate p53. Additionally, MAPK phosphorylation of ASPP2 leads to increased ASPP2 protein levels, suggesting that MAPK can regulate ASPP2 by modulating its protein stability. ASPP1 and ASPP2 deletion fragments were used to examine the regulation of ASPP proteins. Amino-terminus fragments were shown to increase full-length ASPP activity when co-transfected. Moreover, PKA was also found to be a regulator of ASPP2 and was shown to phosphorylate ASPP2 in vitro. Forskolin, a stimulator of PKA, could enhance ASPP2 activity. The results provide the first insight into these novel mechanisms by which ASPP activity may be regulated

Phosphorylation of ASPP2 by RAS/MAPK pathway is critical for its full pro-apoptotic function

We reported recently that apoptosis-stimulating protein of p53 (ASPP) 2, an activator of p53, co-operates with oncogenic RAS to enhance the transcription and apoptotic function of p53. However, the detailed mechanism remains unknown. Here we show that ASPP2 is a novel substrate of mitogen-activated protein kinase (MAPK). Phosphorylation of ASPP2 by MAPK is required for RAS-induced increased binding to p53 and increased transactivation of pro-apoptotic genes. In contrast, an ASPP2 phosphorylation mutant exhibits reduced p53 binding and fails to enhance transactivation and apoptosis. Thus phosphorylation of ASPP2 by RAS/MAPK pathway provides a novel link between RAS and p53 in regulating apoptosis

Diagram summarizes the inter-regulation between ASPP2 and RAS.

<p>ASPP2 binds active RAS at the plasma membrane, thereby increasing RAS signaling to its downstream pathway effectors Raf/MAPK. Activated MAPK phosphorylates ASPP2 which can then relocate to the nucleus and activate p53 pro-apoptotic signaling. </p

Wild-type ASPP2, but not mutant ASPP2 (S827A), translocates to the cytosol and nucleus upon oncogenic RAS activation and this results in an increased interaction with p53.

<p>(<b>A</b>) RAS activation induces cytoplasmic and nuclear translocation of wild-type ASPP2 but not ASPP2 (S827A) in HKe3 ER:HRAS12 cells as detected by immunofluorescence. Arrows indicate cell membrane and stars indicate cytosol. (<b>B</b>) RAS activation enhances the binding of wild-type ASPP2 but not ASPP2 (S827A) to p53. Total cell lysates from HKe3 ER:HRASV12 cells treated with or without 4-OHT were immunoprecipitated with an anti-p53 antibody or control IgG as indicated. </p

Activated Raf enhances the transactivation activity of ASPP2 and p53 to the same extent as activated RAS.

<p>(<b>A</b>) Saos2 cells were transfected as indicated with a Bax-luciferase reporter and the luciferase activity shown. * <i>P</i>=0.05 (<b>B</b>) The value of ASPP2+p53 was taken as 1.0 to reflect the fold increase of ASPP2 and p53 in the presence of activated Raf and mutant RAS. ** <i>P</i>=0.0055; **** <i>P</i>=0.0001.</p

Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR

Somatic activating mutations in EGFR identify a subset of non-small cell lung cancer that respond to tyrosine kinase inhibitors. Acquisition of drug resistance is linked to a specific secondary somatic mutation, EGFR T790M. Here we describe a family with multiple cases of non-small cell lung cancer associated with germline transmission of this mutation. Four of six tumors analyzed showed a secondary somatic activating EGFR mutation, arising in cis with the germline EGFR mutation T790M. These observations implicate altered EGFR signaling in genetic susceptibility to lung cancer

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.</p

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy