eRapa restores a normal life span in a FAP mouse model

Abstract

Mutation of a single copy of the adenomatous polyposis coli (APC) gene results in familial adenomatous polyposis (FAP), which confers an extremely high risk for colon cancer. Apc(Min/+) mice exhibit multiple intestinal neoplasia (MIN) that causes anemia and death from bleeding by 6 months. Mechanistic target of rapamycin complex 1 (mTORC1) inhibitors were shown to improve Apc(Min/+) mouse survival when administered by oral gavage or added directly to the chow, but these mice still died from neoplasia well short of a natural life span. The National Institute of Aging Intervention Testing Program showed that enterically targeted rapamycin (eRapa) extended life span for wild-type genetically heterogeneous mice in part by inhibiting age-associated cancer. We hypothesized that eRapa would be effective in preventing neoplasia and extend survival of Apc(Min/+) mice. We show that eRapa improved survival of Apc(Min/+) mice in a dose-dependent manner. Remarkably, and in contrast to previous reports, most of the Apc(Min/+) mice fed 42 parts per million eRapa lived beyond the median life span reported for wild-type syngeneic mice. Furthermore, chronic eRapa did not cause detrimental immune effects in mouse models of cancer, infection, or autoimmunity, thus assuaging concerns that chronic rapamycin treatment suppresses immunity. Our studies suggest that a novel formulation (enteric targeting) of a well-known and widely used drug (rapamycin) can dramatically improve its efficacy in targeted settings. eRapa or other mTORC1 inhibitors could serve as effective cancer preventatives for people with FAP without suppressing the immune system, thus reducing the dependency on surgery as standard therapy.

Figure 1

Pharmacodynamic data for C57BL/6 mice fed eRapa. (A) Western blots developed with indicated antibodies. C57Bl/6 mice (607–627 days old) were fed eRapa for 42 days, intestine dissected and immunoassayed as described in methods. Segments of the small intestine (proximal and distal) were assayed as indicated. Graphs showing the quantitation of the immunoblot data as measured by the ratio of intensity values for the phosphorylation state-dependent signal (P(240/244)rpS6) to phosphorylation state-independent (rpS6) signal (B)and rpS6 to actin antibody signal (C). (D) Rapamycin blood levels of C57BL/6 mice at 607–627 days of age (42 days on eRapa diets, which averaged 37 ng/ml and 170 ng/ml for the 14 and 42 ppm group, respectively). (E) Rapamycin tissue levels in proximal and distal small intestine of C57BL/6 mice (607–627 days of age and 42 days on diet). Mean levels (μg/g) are: proximal 14 ppm 91.62 ± 14.91, 42 ppm 266.7 ± 26.35; distal 14 ppm 627.3 ± 135.5, 42 ppm 1488 ± 141.6. P values are: 0.001 (proximal 14 ppm compared to 42 ppm); 0.035 (distal 14 ppm compared to 42 ppm); 0.0077 (proximal 14 ppm compared to distal 14 ppm); <0.0001 (proximal 42 ppm compared to distal 42 ppm).

Figure 2

eRapa increases life and health span for Apc^Min/+ mice. (A) eRapa increases life span for Apc^Min/+ mice. The grey box represents a range of median life spans for C57BL/6J female mice taken from previous studies {Wijnhoven et al., 2005, #5122; Yuan et al., 2009, #78681; Zhang et al., 2013, #44400}, which is also representative of syngeneic colonies at the Barshop Institute animal facility used in our study. Note that 60% of the 42 ppm eRapa fed Apc^Min/+ mice have lived beyond this range. (B) Rapamycin blood levels in Apc^Min/+ mice at 217 days (174 days on eRapa diets, which averaged 14 ng/ml or 26.6 ng/ml on 14 ppm or 42 ppm diets, respectively) or at 308 days of age (265 days on eRapa diet, which averaged 10.2 ng/ml or 28.1 ng/ml on 14 ppm or 42 ppm diets, respectively) (C) Polyp count for Apc^Min/+ mice at the time of death. Note the first and third mouse that died from the 42 ppm cohort had no polyps. (D&E) Graph showing quantification of phosphorylation state-dependent signal P(Ser473)Akt to phosphorylation state-independent signal (D) and Akt to actin antibody signal (E). (F) eRapa improves physical activity in Apc^Min/+ mice. Graphed is the mean number of beam breaks (activity) for light (=inactive) and dark (=active) phases of the day. The food area of the cage is excluded. (G) eRapa maintains normal hematocrits in Apc^Min/+ mice. Note the dose response. Also note the normal hematocrit as compared to wild type C57Bl/6 mice (~40%) in the high dose group even at late time points when many wild type C57Bl/6 mice would die from natural causes.

Figure 3

eRapa does not boost tumor growth or tumor-mediated immune suppression. C57BL/6J mice (n=6) were given either Eudragit or 14 ppm eRapa for 3 months and challenged with B16 melanoma. A. Tumor volume. B. Spleen Treg suppression assessed at various CD4⁺responder: Treg ratios. C. Spleen myloid derived suppressor cell (MDSC) suppression assessed at various CD4⁺responder:MDSC ratios. P>0.05 for all in B and C. Treg, regulatory T cell; MDSC, myeloid derived suppressor cell.

Figure 4

eRapa does not reduce mediators of T. gondii immunity during active infection. BL6 mice were infected with 10,000 Me49 strain T. gondii tachyzoites expressing an OVA reporter antigen by intraperitoneal challenge and mice were sacrificed 11 days later. Immune events were acquired from single-cell spleen suspensions on a Becton-Dickinson LSRII. Individual symbols represent individual mice and the bar is the median value. CD69 is an activation marker. Foxp3 is a regulatory T cell marker. CD44⁺CD62L⁻ cells are memory T cells. CD44⁻CD62L⁺ cells are naive T cells. CTRL, mice fed Eudragit control chow. RAPA, mice fed chow with rapamycin 14 ppm. IFNγ, interferon-γ. “Antigen specific” refers to CD8⁺ T cells recognizing the SIINFEKL sequence of the OVA antigen engineered into T. gondii tachyzoites. No differences were statistically significant.