Reduced IL-6 levels and tumor-associated phospho-STAT3 are associated with reduced tumor development in a mouse model of lung cancer chemoprevention with myo-inositol

Abstract

Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer-prone transgenic mice with oncogenic Kras expressed in the airway epithelium (Ccsp^Cre/+ ; Kras^LSL-G12D/+ ) were raised on diets compounded with myo-inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo-inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo-inositol has previously been reported to inhibit phosphoinositide 3-kinase (PI3K) signaling. However, in mice raised on myo-inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL-6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL-6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.

Keywords: IL-6; Kras; chemoprevention; lung cancer; lung cancer chemoprevention; myo-inositol.

Figure 1

A) Lung surface tumor count, performed under 5× magnification, was significantly reduced in mice raised on a diet containing myo-inositol (p=1.04 × 10⁻¹³). B) Bronchial dysplasia (BD) and atypical adenomatous hyperplasia (AAH were reduced in mice raised on myo-inositol (p=0.006 for BD and 0.023 for AAH) C) Representative H&E sections of whole lungs show a gross reduction in dysplastic and neoplastic lesions evident on cancer-prone mice raised on myo-inositol (bottom) than those on a control diet (top). D–F) By Western blot (F) densitometry, oncogenic Kras^G12D (D) levels were reduced in whole lung tissue of mice raised on myo-inositol versus control.Total levels of Kras were unchanged (E) (p=0.005). * indicates p < 0.05 by t-test. n=5 mice for each genotype and diet used for Western blot analyses. β-tubulin loading control in Fig 1F and 2G are identical as same filters are shown.

Figure 2

A–C) Western blot (C) densitometry of phospho-Akt (A) and total Akt (B) levels from mouse whole lungs reveals no significant differences in animals raised on myo-inositol versus those on a regular diet. D–G) Western blot (G) densitometry on mouse whole lungs reveals that levels of phospho-ERK (p-ERK, (D)), total ERK (E), and phospho-cRAF (p-cRAF, (F)) are largely unchanged between CC-LR mice raised on control or myo-inositol diets. There is a mild reduction in total ERK levels in CC-LR mice raised on a myo-inositol diet versus those on control diets, with no significant differences observed in LR mice (p=0.0001). p-cRAF is mildly induced in LR mice raised on a myo-inositol diet (p=0.003).* indicates p < 0.05 by t-test. n=5 mice for each genotype and diet used for Western blot analyses. β-actin loading controls in Fig 2C and 4G are identical as the same filters were used for both figures. Similarly, β-tubulin loading control in Fig 1F and 2G are identical as same filters are shown.

Figure 3

myo-inositol treatment of p53-mutated and Kras-activated mouse lung cancer cells lines (A) 393P, (B) 344SQ, or the Kras-activated p53-wild type mouse lung cancer cell line (C) LKR-13 reveals little reduction in MTS cellular proliferation assays at physiologic (1–10 mM) doses. At higher doses, especially above 50 mM, there is a small reduction in cellular proliferation.

Figure 4

A) Bead-based immunoassays on bronchoalveolar lavage fluid (BALF) reveal a significant decrease in (A) IL-6 and (B) the IL-6 related cytokine LIF in CC-LR mice raised on myo-inositol versus those on a control diet (p=0.053 for LR in IL-6, p=0.019 for CC-LR in IL-6 and 0.036 for CC-LR in LIF). A mild independent induction of IL-6 was noted in LR mice raised on myo-inositol versus those on a control diet. Assays were normalized to total BALF protein concentration as measured by a Bradford assay. C–G) The reduction of IL-6 in CC-LR mice raised on myo-inositol diet is also reflected in decreased levels of phospho-STAT3 (C, p=0.01) and Gp130 (E, p=0.018) seen by whole lung Western blot (G). Also noted was a small induction in p-STAT3 in LR mice raised on myo-inositol (p=0.139). D) Total STAT3 levels are unchanged between CC-LR mice raised on control or myo-inositol diet, where myo-inositol does increase STAT3 protein levels in LR mice (p=0.018). There were no significant changes seen in phospho-STAT1 (F). * indicates p < 0.05 by t-test. n=5 mice for each genotype and diet used for all analyses. β-actin loading controls in Fig 2C and 4G are identical as the same filters were used for both figures.

Figure 5

CC-LR mice show a fall in (A) M1-type antitumoral macrophages and a small rise in (B) protumoral M2-type macrophages. With myo-inositol, there is a recovery of the fall in M1 macrophages with no significant change in M2 macrophages. For M1 macrophages, p=0.002 for LR vs CC-LR on control diet, p=0.03 for LR vs CC-LR on myo-inositol, p=0.008 for CC-LR on control vs myo-inositol diet; for M2 macrophages, p=0.019 for CC-LR vs LR on control diet and p=0.029 for CC-LR vs LR on myo-inositol. C) myo-inositol at 5 mM does not affect healthy PBMC differentiation into an M1-like phenotype (CD68^hiCD86⁺CD163⁻) but increases M2-like differentiation (CD68^hiCD86⁺CD163⁺). In M2 differentiation media, PBMCs that do not become CD163⁺ show an M2b-like phenotype. The percentage of these cells decrease with myo-inositol. D–F) myo-inositol decreases IL-6 secretion in M1-like (D, p=0.010), M2-like (E, p=0.001), and M2b-like (F, p=0.022) cells. * indicates p < 0.05 by t-test. n=5 mice for each genotype and diet used.

Figure 6

A) Immunohistochemical staining for p-STAT3 shows a rise in levels in CC-LR mice that is significantly decreased on a myo-inositol diet (p=0.023). B) p-STAT3 levels within tumors are higher, and are significantly reduced in CC-LR mice treated with myo-inositol (p=2.21 × 10⁻⁶). C) Immunohistochemistry shows reduced pSTAT3 staining in adenomas from mice raised on myo-inositol versus control diets Scale bar = 200 μm, micrographs at 10× magnification with a 20× inset. n=4 mice for each diet and genotype used for immunohistochemical analyses.