Selective activation of p53-mediated tumour suppression in high-grade tumours

Abstract

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10-15%. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras. p53 is also frequently inactivated in NSCLC and, because oncogenic Ras can be a potent trigger of p53 (ref. 3), it seems likely that oncogenic Ras signalling has a major and persistent role in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease. Here we model the probable therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras. Surprisingly, p53 restoration failed to induce significant regression of established tumours, although it did result in a significant decrease in the relative proportion of high-grade tumours. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked upregulation in Ras signal intensity and induction of the oncogenic signalling sensor p19(ARF)( )(ref. 6). Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and in the efficacy of therapeutic p53 restoration to eradicate cancers.

Figure 1. Heterogeneous therapeutic impact of p53 restoration in Kras^G12D driven lung tumours

a. Schematic representation of the experimental treatment regime. Kras^G12D was activated in the lung epithelium of 8 week old KR;p53^KI/KI mice by adenoviral-Cre nasal inhalation and the resulting tumours treated with Tam or vehicle (Ctrl) 15 weeks after adenoviral infection. b. Haematoxylin and Eosin staining of lung sections from KR;p53^KI/KI mice showing tumour load after 7 daily control (Ctrl) or Tam treatments. c. Quantification of Ki67 positive cells per lung tumour from 7 day Tam/Ctrl-treated KR;p53^KI/KI mice. Error bars indicate standard error of mean (Ctrl: s.e.m=1.20 n=55; Tam: s.e.m=1.31 n=37). * P=0.0003, Student’s t-test. d. Percent of apoptotic (TUNEL-positive) tumours (scored as a minimum of 1 positive cell per tumour section) in 7 day Ctrl and Tam treated KR;p53^KI/KI lungs (n=37 Ctrl; n=22 Tam treated tumours). * P=0.0064, Pearson Chi square. e. KR;p53^KI/KI lung tumours from KR;p53^KI/KI treated for 6 hrs with Tam, showing either no discernible TUNEL staining (Neg) or significant levels of TUNEL staining (Pos). Scale bar=100 µm.

Figure 2. Heterogeneous p53 activation and p19^ARF up-regulation in KR;p53^KI/KI tumours

a. MicroCT-derived plots depicting changes in tumour volume during a 7-day treatment. 10 independent tumours are shown before (day 0) and after (day 7) daily Tam (red lines, filled symbols) or sham (black lines, open symbols) treatments. b. Taqman analysis of CDKN1A expression in individual laser-captured lung tumours from KR;p53^KI/KI mice treated for 7 days with vehicle (black circles) or Tam (red squares). Tumours were harvested 24 hrs after the final Ctrl/Tam treatment. Where indicated (IR +, left panel) mice were exposed to a single dose of γ-radiation 2 hrs after the last Tam/Ctrl treatment. Each circle/square represents a single tumour. c. IHC data comparing levels of p19^ARF expression in low and high-grade tumours as well as in transitional lesions exhibiting both low and high-grade features. Scale bars=50 µm. d. Co-immunostaining for p19^ARF and p21^cip1 in KR;p53^KI/KI lung tumours from mice treated for 6 hrs with Tam. Representative fields shown, one at low (upper panel) and one at high magnification (lower panel). Scale bar=50 µm.

Figure 3. p53 restoration targets high-grade, but not low-grade, lung tumour cells

a. Co-immunostaining for p19^ARF and the proliferation marker Ki67 in lung tumours from KR;p53^KI/KI mice treated for 24 hrs with vehicle (Ctrl, upper row) or Tam (four lower rows). Row 2 and 3 illustrate the profound anti-proliferative impact (low Ki67) of p53 restoration in tumours with high p19^ARF levels. By contrast, the lower two rows show lack of growth inhibition following p53 restoration in tumours lacking detectable p19^ARF. Scale bar = 50 µm. b. Quantification of low versus high-grade tumour frequencies in lungs of KR;p53^KI/KI mice treated for 7 days with either vehicle (Ctrl) or Tam (n=143 Ctrl; n=163 Tam). P=0.0001, Pearson Chi square. c. Representative images show IHC for BrdU in high-grade tumours from 7-day treated Ctrl (Ctrl, upper panel) or Tam mice (lower panel). BrdU was administered 2 hrs before harvesting. Arrows highlight high-grade cells in each tumour (filled, BrdU positive and open, BrdU negative). Scale bars=50 µm.

Figure 4. High-grade lung tumours exhibit increased Kras signalling

a. IHC for p19^ARF and p-ERK in consecutive sections of three independent low-to-high-grade transition tumours from KR;p53^KI/KI mice. Scale bar = 200 µm. b. Kras allele analysis was performed on genomic DNA from KR;p53^KI/KI lung tumours following laser capture microdissection. p-ERK IHC was used to define areas of low, mixed or high p-ERK (upper panel, Scale bar=50µm) and consecutive slides used for LCM of defined regions (see dotted areas). DNA was isolated from LCM material and the Kras genomic region amplified by PCR and digested with HindIII (lower panel). For each tumour, the undigested (−) and digested (+) PCR fragments were run alongside and the wt (Kras, higher band) and mutant alleles (G12D, lower band) are indicated. Control lung tissue from heterozygous (Kras^G12D/+: Ctrl) and wild-type (WT) mice was also analyzed.