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. 2010 Mar 18;29(11):1588-97.
doi: 10.1038/onc.2009.452. Epub 2009 Dec 7.

TSC1 loss synergizes with KRAS activation in lung cancer development in the mouse and confers rapamycin sensitivity

M-C Liang  1 , J Ma, L Chen, P Kozlowski, W Qin, D Li, J Goto, T Shimamura, D N Hayes, M Meyerson, D J Kwiatkowski, K-K Wong
Affiliations

TSC1 loss synergizes with KRAS activation in lung cancer development in the mouse and confers rapamycin sensitivity

M-C Liang et al. Oncogene. .

Abstract

Germline TSC1 or TSC2 mutations cause tuberous sclerosis complex (TSC), a hamartoma syndrome with lung involvement. To explore the potential interaction between TSC1 and KRAS activation in lung cancer, mice in which Tsc1 loss and Kras(G12D) expression occur in a small fraction of lung epithelial cells were generated. Mice with a combined Tsc1-Kras(G12D) mutation had dramatically reduced tumor latency (median survival: 11.6-15.6 weeks) in comparison with Kras(G12D) alone mutant mice (median survival: 27.5 weeks). Tsc1-Kras(G12D) tumors showed consistent activation of mTOR (mammalian target of rapamycin)C1 and responded to treatment with rapamycin, leading to significantly improved survival, whereas rapamycin had minor effects on cancers in Kras(G12D) alone mice. Loss of heterozygosity for TSC1 or TSC2 was found in 22% of 86 human lung cancer specimens. However, none of the 80 lung cancer lines studied showed evidence of the lack of expression of either TSC1 or TSC2 or a signaling pattern corresponding to complete loss. These data indicate that Tsc1 loss synergizes with the Kras mutation to enhance lung tumorigenesis in the mouse, but that this is a rare event in human lung cancer. Rapamycin may have unique benefit for patients with lung cancer, for whom the TSC1/TSC2 function is limited.

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Figures

Figure 1
Figure 1. Loss of Tsc1 markedly accelerates oncogenic Kras-driven lung tumorigenesis in the mouse, with activation of mTORC1
(A) LSL-KrasG12D Tsc1L/L mice display accelerated lung cancer development and early mortality. Kaplan-Meier survival curves of mouse cohorts following nasal inhalation of 5 × 106 PFU of AdCre. The median survival was 11.6 weeks for LSL-KrasG12D Tsc1L/L mice, 15.6 weeks for LSL-KrasG12D Tsc1L/+ mice, 27.5 weeks for LSL-KrasG12D mice, and over a year for Tsc1L/L mice. (B) H&E-stained sections of lung from mice 12.5 weeks following AdCre inoculation shows extensive tumor in both LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ mice. Low power (left) and high power (right) views are shown. Bar, 100μm. (C) Immunoblot analysis of protein expression and mTOR signaling in tumors from these mice shows that Tsc1 and Tsc2 expression is reduced in the lung tumor nodules of both LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ mice in comparison to tumors from LSL-KrasG12D mice. pS6(S240/244) expression is increased and pAKT(S473) expression is reduced, consistent with mTORC1 activation in these tumors. pERK levels are higher in the LSL-KrasG12D mice, compared to other genotypes. AKT, S6, ERK, and actin are all controls. Note that one lung tumor sample was depleted, and was not included in the Tsc2 blot, indicated by *. (D) MLPA analyses of Tsc1 alleles demonstrate conversion of L to K alleles in both LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ tumor nodules. Yellow, red, and blue bars indicate the relative amount of wild type (W), knockout (K), and conditional allele (L), respectively. The four samples at right (a-d) are control samples from mice with genotypes KW, KW, LW, and LL, respectively. Note that the W allele persists in LSL-KrasG12D Tsc1L/+ tumor nodules.
Figure 2
Figure 2. MLPA analyses of human lung cancers identifies TSC1 and TSC2 genomic loss
(A) Summary of MLPA analyses on 86 human NSCLC specimens demonstrates the proportion of samples with genetic events affecting TSC1 or TSC2. (B) MLPA graphs. a, NSCLC sample without TSC1 or TSC2 loss; b, sample with TSC1 genomic loss, with 60% signal for all TSC1 probes; c, sample consistent with complete genomic deletion of TSC1, with 40% signal for 8 of 10 probes within TSC1; d, MLPA analysis of a control sample from a TSC patient with a heterozygous germline deletion involving exons 8 - 23.
Figure 3
Figure 3. Rapamycin decreases tumor burden and causes apoptosis in LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ mice
(A) MRI images (left and middle columns) of mice before and after 2 weeks of rapamycin treatment (6 mg/kg IP every other day). H indicates the heart. H&E stained lung tissue sections (right column) after 2-5 weeks of rapamycin treatment show evidence of regression in the LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ mice. The top three H&E sections were from mice treated for 2 weeks, the bottom mouse was treated for 5 weeks. Bar, 100μm. (B) Representative photographs of cancers from the LSL-KrasG12D Tsc1L/L and LSL-KrasG12D mice showing apoptosis (TUNEL staining) without treatment, and after two doses of rapamycin. Bar, 100μm.
Figure 4
Figure 4. Quantitative tumor response, apoptosis, proliferation, and pS6 expression in response to rapamycin in LSL-KrasG12D Tsc1L/L, LSL-KrasG12D, and other mice
(A) Relative change in tumor burden in mice treated with rapamycin for 2 weeks (circles). MRI scans from before and after treatment were assessed for tumor volume in mm3 by a blinded observer, and the ratio between after treatment and before treatment volumes was plotted. Six mice of various genotypes were placebo-treated controls in this experiment, shown as squares. The lines indicate average change among the treated mice. (B) Scatter plots of number of positive cells per high power field (HPF) in lung tumor nodules stained to show evidence of apoptosis (TUNEL). The mice had genotypes LSL-KrasG12D Tsc1L/L or LSL-KrasG12D, and were treated with rapamycin 6mg/kg every other day for 0, 1, 2, or 4 doses, as indicated on the x axis. (C) Scatter plots of number of positive cells per HPF in lung tumor nodules stained to show evidence of proliferation (antibodies against Ki67), as in B. (D) Scatter plots of relative staining intensity for pS6(S240/244) in tumor nodules from rapamycin-treated mice, as in B. For B-D, all pairwise comparisons between LSL-KrasG12D Tsc1L/L and LSL-KrasG12D tumors after the same number of doses are not significant (P > 0.05 by Mann Whitney test), except for the comparison between TUNEL positivity (B) after 2 and 4 doses, p=0.028 and 0.048 respectively.
Figure 5
Figure 5. Rapamycin improves survival of LSL-KrasG12D Tsc1L/L and LSL-KrasG12D Tsc1L/+ mice
Kaplan-Meier survival curves of mice of two genotypes treated with either rapamycin (6 mg/kg IP every other day) or placebo, beginning 9 weeks following AdCre inhalation. Rapamycin significantly extended the survival of both LSL-KrasG12D Tsc1L/L (p=0.0018) and LSL-KrasG12D Tsc1L/+ mice (p=0.002).
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