Mad2-induced chromosome instability leads to lung tumour relapse after oncogene withdrawal

Abstract

Inhibition of an initiating oncogene often leads to extensive tumour cell death, a phenomenon known as oncogene addiction. This has led to the search for compounds that specifically target and inhibit oncogenes as anticancer agents. However, there has been no systematic exploration of whether chromosomal instability generated as a result of deregulation of the mitotic checkpoint pathway, a frequent characteristic of solid tumours, has any effect on oncogene addiction. Here we show that induction of chromosome instability by overexpression of the mitotic checkpoint gene Mad2 in mice does not affect the regression of Kras-driven lung tumours when Kras is inhibited. However, tumours that experience transient Mad2 overexpression and consequent chromosome instability recur at markedly elevated rates. The recurrent tumours are highly aneuploid and have varied activation of pro-proliferative pathways. Thus, early chromosomal instability may be responsible for tumour relapse after seemingly effective anticancer treatments.

Figure 1. Mad2 Overexpression Cooperates with Kras^G12D in Lung Tumorigenesis

a, Mad2 western blot of TI-KM lungs maintained with (+) or without (−) doxycycline. b, Lung weights from TI-K and TI-KM mice on doxycycline for 8 weeks (Right). Representative macroscopic pictures show lung tumor-size (dotted lines). c, Total tumor area in TI-K and TI-KM lungs. d, H&E from TI-K or TI-KM mice after 8 weeks on doxycycline (left panel), Ki67 staining (middle panel) and TUNEL (right panel) (black bar: 100 μm). e, Tumor nodule/mm2 of lung tissue in TI-K versus TI-KM animals. f, Percentage of Ki67 positive cells. g, Kaplan-Meier curve of TI-K and TI-KM mice. Error bars represent mean and s.e.m. from at least 4 different mice. P values were determined by unpaired t test.

Figure 2. Mad2 Overexpression Increases Aneuploidy but is not Enough to Overcome Oncogene Addiction

a, FISH images from TI-M normal lung, TI-K and TI-KM lung tumor showing aneuploid cells in yellow and normal cells in white. DNA: blue, chromosome 12 probe (green), 16 (red) and 17 (orange). b, Frequency of aneuploidy in 5 individual mice from each group. c, MR images and histological sections of lung tumors from TI-K and TI-KM mice after 18 weeks on doxycycline (left). After 2 weeks of doxycycline withdrawal, lung opacities disappeared from MR images, and tumors regressed histologically. H: heart, T: tumor. Scale bar: 200 μm.

Figure 3. Chromosomal Instability Increases the Likelihood of Recurrence in Kras Tumors

a, MRI from TI-K and TI-KM mice on doxycycline showing lung tumors (yellow circles) (left panel), after 2-6 weeks of doxycycline withdrawal showing complete regression (middle panel) and after 14-16 weeks off doxycycline (right panel) showing recurrent tumors in TI-KM mice at the same anatomical site as the primary tumor. Histological sections: right panel. b, Relapse-free survival curve of TI-K and TI-KM mice; censured events indicated by circles. c, Initial tumor volume of TI-K and TI-KM mice that never recur and TI-KM that developed a relapse. d, RT-PCR from normal, TI-K and TI-KM lungs on doxycycline showing the corresponding transgenes and from TI-K, TI-KM and relapses off doxycycline. e, SP-C, Ki67 and HA immunostaining of a relapse. (Black bar: 200 μm, blue bar: 100 μm).

Figure 4. Recurrent Tumors from TI-KM Mice are Heterogeneous and Highly Aneuploid

a, FISH images from a TI-K and TI-KM regressed lung and a TI-KM tumor relapse showing aneuploid cells in yellow lines and normal cells in white. b, Frequency of aneuploidy in 3 independent TI-K and 3 TI-KM regressed lungs and 3 recurrent tumors of TI-KM mice showing an increase in aneuploid cells in the recurrent tumors. P values were determined by unpaired t test. c, Immunohistochemistry of pERK, pStat3, pAKT and pS6 in tumor relapses showing positive staining for all 3 pathways in relapses 1,3 and 8 while relapses 4 and 7 are only positive for pAKT and pS6. Scale bar: 100 μm.