Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of β-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTOR and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression.

Intestinal inflammation is associated with low levels of mucosal ATP, highlighting the importance of mitochondrial function associated with ATP production in the pathophysiology of the disease. In the inflamed colon of humans and mice, we found decreased levels of mitochondrial complex cytochrome oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ cells with reduced mitochondrial function linked to ATP production by selective depletion of mitochondrial DNA. In these cells, RNA sequencing revealed a substantial number of differentially expressed transcripts, among which 240 belonged to inflammatory pathways activated in human inflamed colon and TNF-α-treated cells (false discovery rate < 0.05). TNF-α treatment of colonic ρ cells augmented IL-8 expression by 9-fold ( < 0.01) NF-κB compared to TNF-α-treated control. Moreover, reduced mitochondrial function facilitated TNF-α-mediated NF-κB luciferase promoter activity as a result of lowered inhibitory IκBα (nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor, α), leading to elevated NF-κB. In cells with reduced mitochondrial function, TNF-α facilitated AMPKα2 activation by 8-fold ( < 0.01), which was involved in NF-κB-dependent IL-8 expression. Last, in human and mouse colon, anti-TNF-α treatment restored reduced mitochondria-dependent inflammation. We propose that selective targeting of this novel mechanism provides new treatment opportunities for intestinal inflammation.-Heller, S., Penrose, H. M., Cable, C., Biswas, D., Nakhoul, H., Baddoo, M., Flemington, E., Crawford, S. E., Savkovic, S. D. Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

Background & AimsDiminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth.MethodsFOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3).ResultsIn mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth.ConclusionsWe identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

Intestinal inflammation has been recently characterized by the dysregulation of lipids as metabolic and energy sources, revealing a novel feature of its pathophysiology. Because intracellular lipids, stored in dynamic lipid droplets (LDs), provide energy for cellular needs, we investigated whether they play a role in intestinal inflammation. In the inflamed intestine of mice, elevated LDs were found in colonic and infiltrating immune cells as shown by staining for the LD coat protein PLIN2 and for lipids with BODIPY. In colonic cells, TNF stimulated LD increases by receptor signaling rely on phosphatidylinositol 3-kinase activation. Downstream, TNF triggered a negative regulatory loop between LDs and the transcription factor FOXO3. This was shown in the colon of Foxo3-deficient mice, where elevation in PLIN2 and lipids were further facilitated by inflammation and were more prominent relative to wild-type, whereas, in colonic cells, inhibition of lipogenesis blocked the TNF-mediated loss of FOXO3. Furthermore, blockade of PGE2 synthesis abrogated TNF-stimulated increases in LDs and FOXO3 inactivation. We found in colonic tissue of Foxo3-deficient mice higher levels of cyclooxygenase-2, a mediator of prostaglandin E2 (PGE2) synthesis, supporting involvement of PGE2 in the LD-FOXO3 regulatory loop. Ultimately, TNF-stimulated lipogenesis leading to elevated LDs facilitated NF-κB-mediated increases in IL-8 protein, which is associated with the surface of LDs found in the lumina of the endoplasmic reticulum and Golgi apparatus. This novel immunometabolic mechanism of colonic inflammation involving elevated LDs could provide opportunities for new treatment options.

BackgroundMitochondrial reprogramming has emerged as a hallmark of cancer pathobiology. Although it is believed this reprogramming is essential for cancer cells to thrive, how it supports cancer pathobiology is unclear. We previously generated colonic ρ0 (rho0) cells with reduced mitochondrial energy function and acquired their transcriptional signature. Here, we utilized a bioinformatics approach to identify their changes linked to cancer pathobiology.MethodsHuman colon cancer HCT116 cells, control and ρ0, were used for qPCR. Bioinformatics analysis: GeneCards, Kaplan-Meier Survival, GENT, cBioPortal.ResultsThe colonic ρ0 transcriptome was linked with proliferation, DNA replication, survival, tumor morphology, and cancer. Among differentially expressed transcripts, 281 were regulators or biomarkers of human colon cancer especially those with inflammatory microsatellite instability (MSI). We identified and validated novel transcripts in ρ0 cells with altered expression in human colon cancer. Among them DGK1, HTR7, FLRT3, and ZBTB18 co-occurred with established regulators of human colon cancer pathobiology. Also, increased levels of DGKI, FLRT3, ZBTB18, and YPEL1 as well as decreased levels of HTR7, and CALML6 were linked to substantially poorer patient survival.ConclusionWe identified established and novel regulators in colon cancer pathobiology that are dependent on mitochondrial energy reprogramming and linked to poorer patient survival.

Opioid dependence can be difficult to manage using existing pharmacotherapies. A long-acting opioid with low abuse liability that substitutes for a shorter-acting opioid may improve treatment of opioid use disorders (OUDs). We recently characterized an endomorphin (EM) analog (ZH853) that produced a longer duration of antinociception compared with morphine, but did not produce self-administration or several other adverse effects preclinically. Here, we further characterized ZH853 in tests of antinociception, abuse liability, and drug discrimination. A conditioned place preference (CPP) procedure, that included a locomotor activity assessment, was used to test abuse liability in rats. Subsequently, dopamine (DA) cell-somas located in the ventral tegmental area (VTA) from these rats were assessed by size using immunohistochemistry and Stereo Investigator software. A hot-plate antinociception test in male and female mice confirmed central penetration. Morphine-substitution effects of several EM analogs (ZH850, ZH831, and ZH853) were tested in a drug discrimination (DD) procedure in rats. Morphine produced dose-dependent CPP and locomotor sensitization and reduced the size of DA cell somas in VTA, whereas ZH853 did not produce any of these effects relative to control. The antinociceptive effects of ZH853 were m-receptor selective since b-funaltrexamine antagonized these effects. Rats responded on a morphinetrained lever when injected with ZH831 and ZH853 during DD experiments. The favorable morphine-substitution effects of these EM analogs relative to their low abuse liability indicate promising novel compounds that may improve treatment of OUD. SIGNIFICANCE STATEMENT In this experiment, we investigated the preclinical effects of novel endomorphin analogs for use as substitution therapies for opioid use disorder, a problem that has contributed to an opioid overdose epidemic. Several endomorphin analogs substituted for morphine without producing adverse effects, including reward behaviors associated with abuse liability. These compounds have the potential to become important additional tools to treat opioid use disorders.

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