Long-term treatment of cancer-prone germline PTEN mutant mice with low-dose rapamycin extends lifespan and delays tumour development

Abstract

PTEN is one of the most commonly inactivated tumour suppressor genes in sporadic cancer. Germline heterozygous PTEN gene alterations also underlie PTEN hamartoma tumour syndrome (PHTS), a rare human cancer-predisposition condition. A key feature of systemic PTEN deregulation is the inability to adequately dampen PI3-kinase (PI3K)/mTORC1 signalling. PI3K/mTORC1 pathway inhibitors such as rapamycin are therefore expected to neutralise the impact of PTEN loss, rendering this a more druggable context compared with those of other tumour suppressor pathways such as loss of TP53. However, this has not been explored in cancer prevention in a model of germline cancer predisposition, such as PHTS. Clinical trials of short-term treatment with rapamycin have recently been initiated for PHTS, focusing on cognition and colon polyposis. Here, we administered a low dose of rapamycin from the age of 6 weeks onwards to mice with heterozygous germline Pten loss, a mouse model that recapitulates most characteristics of human PHTS. Rapamycin was well tolerated and led to a highly significant improvement of survival in both male and female mice. This was accompanied by a delay in, but not full blockade of, the development of a range of proliferative lesions, including gastro-intestinal and thyroid tumours and endometrial hyperplasia, with no impact on mammary and prostate tumours, and no effect on brain overgrowth. Our data indicate that rapamycin may have cancer prevention potential in human PHTS. This might also be the case for sporadic cancers in which genetic PI3K pathway activation is an early event in tumour development, such as endometrial cancer and some breast cancers. To the best of our knowledge, this is the first report of a long-term treatment of a germline cancer predisposition model with a PI3K/mTOR pathway inhibitor. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: PHTS; PI 3-kinase; PTEN; cancer prevention; drug; hamartoma; kinase inhibitor; mTORC1; rapamycin; rare disease; syndrome.

Figure 1

Long‐term treatment with low dose rapamycin improves overall survival in Pten ^+/− mice. (A) Effect of rapamycin on 4T1 tumour growth. BALB/c mice were injected with 4T1 mammary cancer cells and treated with rapamycin IP (8 mg/kg) (once a day – 5 days on/2 days off) or with the eRapa diet. Mice injected with vehicle (IP vehicle control) or on the control diet were used as controls. Tumours were measured using calipers. (B) Immunoblot analysis of protein extracts from the livers of mice treated as indicated. Statistical analysis was performed using a Mann–Whitney test. **p < 0.01. (C) Plasma rapamycin concentrations in Pten ^+/− and Pten ^+/+ mice on a mixed (C57BL/6J × Sv129) background fed the control or eRapa diet from the age of 6 weeks. Blood samples were taken at 6 months of age (18 weeks after diet switch). The table shows the levels of rapamycin observed at peak levels (blood sample taken at 9 am) and trough levels (blood sample taken at 2 pm, after 6 h of starvation). (D) Kaplan–Meier survival curves for female Pten ^+/− mice on a mixed (C57BL/6J × Sv129) background fed the control or eRapa diet from the age of 6 weeks. Mice were monitored and were euthanised for welfare reasons (masses with a combined size of ≥1.4 cm² surface area or ill health) or at the end of the study. (E) Same as in D, but for male mice. Statistical analysis in D and E was performed using the log‐rank (Mantel–Cox) test and Gehan–Breslow–Wilcoxon test. ****p < 0.0001. (F, G) Histopathological analysis was carried out on all mice to assess the incidence of specific tumour types in female (F) and male (G) Pten ^+/− mice fed the control or eRapa diet from the age of 6 weeks onwards. Statistical analysis was performed using Fisher's exact test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

Low dose rapamycin is effective at delaying GI tract, thyroid, and adrenal tumours, and lymphoid hyperplasia. Pten ^+/− mice on a mixed (C57BL/6J × Sv129) background were fed the control or eRapa diet from the age of 6 weeks onwards. Mice were euthanised for welfare reasons (masses with a combined size of ≥1.4 cm² surface area or ill health) or at the end of the study, and histopathological analysis was performed. (A) Incidence of GI tumours and distribution of age of mice presenting with the indicated tumour in female (left panel) and male (middle panel) mice. Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). Representative photomicrographs of H&E‐stained sections showing GI tract histopathology in Pten ^+/− and Pten ^+/+ mice on the control or eRapa diet (right panel). (B) Incidence of thyroid tumours and distribution of age of mice presenting with the indicated tumours in female (left panel) and male (middle panel) mice. Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). Representative photomicrographs of H&E‐stained sections showing thyroid histopathology in Pten ^+/− and Pten ^+/+ mice on the control or eRapa diet (right panel). (C) Grade of lymphoid hyperplasia (top panel) and representative photomicrographs of H&E‐stained sections showing lymph node histopathology in Pten ^+/− and Pten ^+/+ mice on the control or eRapa diet (bottom panel). (D) Incidence and distribution of age of mice presenting with phaeochromocytoma in female (top left panel) and male (top right panel) mice. Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). Representative photomicrographs of H&E‐stained sections showing the histopathology of the adrenal glands in Pten ^+/− and Pten ^+/+ mice on the control or eRapa diet (bottom panel). Statistical analysis was performed using Mann–Whitney tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bars in A, B, C and D represent 100 μm.

Figure 3

Low dose rapamycin is effective at delaying endometrial hyperplasia. Pten ^+/− mice on a mixed (C57BL/6J × Sv129) background were fed the control or eRapa diet from the age of 6 weeks onwards. Mice were euthanised for welfare reasons (masses with a combined size of ≥1.4 cm² surface area or ill health) or at the end of the study, and histopathological analysis was performed. (A) Incidence and age distribution of endometrial proliferative lesions in mice presenting with the indicated lesions in female Pten ^+/− mice on the control or eRapa diet. Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). (B) Grade of atypical hyperplasia seen in mice in the life‐long study in A, determined using the Milam scoring system (described in supplementary material, Supplementary materials and methods). (C) Representative photomicrographs of H&E‐stained sections showing the uterine histopathology in Pten ^+/− and Pten ^+/+ mice on the control or eRapa diet. (D) Rapamycin Timed Study I: Pten ^+/− mice at 6 weeks of age were switched to the control or eRapa diet and sacrificed when 6 months old, followed by analysis of the uteri by H&E staining and Milam scoring. (E) Representative examples of IHC performed on uteri from 4‐ and 6‐week‐old female Pten ^+/+ and Pten ^+/− mice showing loss of PTEN immunoreactivity in endometrial glands of Pten ^+/− mice at 6 weeks (indicated by black arrowheads) but not at 4 weeks of age. Additional images are shown in supplementary material, Figure S2. (F) Rapamycin Timed Study II: Pten ^+/− mice at 4 weeks of age were switched to the control or eRapa diet and sacrificed at the age of 6 months, followed by analysis of uteri by H&E staining and Milam scoring. (G) Representative examples of IHC performed on uteri from female mice from Timed Study II showing loss of PTEN immunoreactivity in endometrial glands of Pten ^+/− mice on the control or eRapa diet (indicated by black arrowheads), with a corresponding increase in pAKT‐S473 immunoreactivity within the same glands (indicated by black arrowheads). Additional images are shown in supplementary material, Figure S3. Statistical analysis was performed using Mann–Whitney tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scale bars in C, E and G represent 100 μm.

Figure 4

Low dose Rapamycin is not effective at delaying prostate and mammary tumours and does not rescue macrocephaly. Pten ^+/− mice on a mixed (C57BL/6J × Sv129) background were fed the control or eRapa diet from the age of 6 weeks onwards. Mice were euthanised for welfare reasons (masses with a combined size of ≥1.4 cm² surface area or ill health) or at the end of the study, and histopathological analysis was performed. (A) Incidence of mammary tumours and age distribution of female Pten ^+/− mice presenting with the indicated lesions (left panel). Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). Representative photomicrographs showing the histological characteristics of mammary tumours (right panel). (B) IHC analysis of pAKT‐S473 in mammary tissues of 6‐month‐old female mice switched to the control or eRapa diet at 4 weeks of age. No signal for pAKT‐S473 was observed in mammary tissues of mice on the control or eRapa diet. Staining for pAKT‐S473 in endometrial tissue of the same mouse processed on the same slide is shown as an inset. Note the positive pAKT‐S473 staining in Pten ^+/− endometrium but absence of the staining in Pten ^+/+ endometrium. Additional images are shown in supplementary material, Figure S4. (C) Incidence of prostate tumours in male mice (top panel). Dotted lines show the median survival age of mice on the control diet (orange) or the eRapa diet (green). Representative photomicrographs showing the histological characteristics of prostate tumours (bottom panel). (D) Brain weight of Pten ^+/− and Pten ^+/+ littermate mice fed the control or eRapa diet from 6 weeks of age for 6 or 18 weeks. Statistical analysis was performed using Mann–Whitney tests. Scale bars in A, B and C represent 100 μm.

Figure 5

Effects of low dose rapamycin treatment on glucose metabolism. Pten ^+/− mice and littermate Pten ^+/+ mice were fed the control or eRapa diet from 6 weeks of age. (A, B) ITT and (C, D) GTT assays performed at the indicated time points. Bar graphs show measurements of the area under the curve (AUC). (E) Mice were starved for 6 h and their fasting blood glucose was measured. (F) Fed blood glucose measurements, taken at 9 am at the indicated time points. (G) Insulin levels in blood samples harvested at 9 am from 6‐month‐old mice. Statistical analysis was performed using Mann–Whitney tests. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.