Apc inactivation, but not obesity, synergizes with Pten deficiency to drive intestinal stem cell-derived tumorigenesis

Abstract

Obesity is a major risk factor for colorectal cancer and can accelerate Lgr5+ intestinal stem cell (ISC)-derived tumorigenesis following inactivation of Apc. However, whether non-canonical pathways involving PI3K-Akt signaling in ISCs can lead to tumor formation, and if this can be further exacerbated by obesity is unknown. Despite the synergy between Pten and Apc inactivation in epithelial cells on intestinal tumor formation, their combined role in Lgr5+-ISCs, which are the most rapidly dividing ISC population in the intestine, is unknown. Lgr5+-GFP mice were provided low-fat diet (LFD) or high-fat diet (HFD) for 8 mo and the transcriptome was evaluated in Lgr5+-ISCs. For tumor studies, Lgr5+-GFP and Lgr5+-GFP Ptenflox/flox mice were tamoxifen treated to inactivate Pten in ISCs and provided LFD or HFD until 14–15 mo of age. Finally, various combinations of Lgr5+-ISC specific, Apc and Pten-deleted mice were generated, and evaluated for histopathology and survival. HFD did not overtly alter Akt signaling in ISCs, but did increase other metabolic pathways. Pten deficiency, but not HFD, increased BrdU positive cells in the small intestine (P<0.05). However, combining Pten and Apc deficiency synergistically increased proliferative markers, tumor pathology and mortality, in a dose-dependent fashion (P<0.05). In summary, we show that HFD alone fails to drive Akt signaling in ISCs and that Pten deficiency, is dispensable as a tumor suppressor in Lgr5+-ISCs. However, combining Pten and Apc deficiency in ISCs synergistically increases proliferation, tumor formation, and mortality. Thus, aberrant Wnt/β-catenin, rather than PI3K-Akt signaling, is requisite for obesity to drive Lgr5+ISC-derived tumorigenesis.

Dietary Walnuts Protect Against Obesity-Driven Intestinal Stem Cell Decline and Tumorigenesis

“…However, we observed a contrasting result, whereby obesity impaired ISC function that was preserved by walnuts. While a definitive explanation of these disparate results is not entirely clear, an important distinction between our approach ( 36 ) and others ( 9 ), is that our HFD-fed animals are compared to a carefully-matched, low fat purified diet, rather than a poorly-defined chow-based formula. The contrasting compositions of purified vs. chow based formulations, and the ability of multiple individual components in chow diet to impact intestinal function and tumorigenesis, independent of calories, has been extensively discussed elsewhere ( 36 , 74 ).…”

“…Evaluation of the gastrointestinal tract was performed following isolation and division into four segments: duodenum, jejunum, ileum and colon, as previously described ( 6 , 36 ). Tissues were rolled and fixed overnight in 10% neutral-buffered formalin at 4°C, processed through a series of alcohols and xylenes, and embedded in paraffin.…”

“…Total RNA from frozen tissues was isolated using the Trizol procedure as described previously ( 36 ). In brief, first-strand complementary DNA (cDNA) was synthesized with random primers and total RNA as a template using Biorad iScript cDNA Synthesis Kit.…”

“…Plasma free fatty acids (Wako Diagnostics, Richmond, VA, USA) and triglycerides (Sigma, St. Louis, MO) were analyzed using standard calorimetric assays. Plasma insulin levels were measured by a rat/mouse ELISA (EMD Millipore, Inc.) with rat insulin standards using a spectrophotometer (Biorad iMark platereader) following the manufacturer's instructions ( 36 , 37 ). In addition, a Bio-Plex MAGPIX Multiplex Reader (Biorad Inc., Hercules, CA) was used to measure the following 23 inflammatory mediators simultaneously in plasma: Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC, LIF, LIX, M-CSF, MCP-1, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, and VEGF.…”

“…In order to assess intestinal stem cell (ISC) function, crypts were isolated from the small intestine of LFD, HFD and HFD+W fed mice ( n = 4 group) following 4 weeks on diet, as previously described ( 36 , 42 ). Isolated crypts were washed with ADF medium, centrifuged at 800 rpm for 5 min, re-suspended in ADF medium and counted on a hemocytometer.…”