Sarah P. Short

Although colorectal cancer is the second leading cause of cancer-related mortality in the United States, there has been limited progress in recent years in identifying new therapeutic options. However, cancer cells have been shown to be sensitive to ferroptosis, an iron-dependent lipid peroxide–induced form of cell death. In this issue of Cancer Research, DeAngelo and colleagues aimed to better understand the mechanisms underlying ferroptosis in colorectal cancer. However, using the ferroptosis-inducing small molecule RAS-selective lethal 3 (RSL3), they observed effects on colorectal cancer cells independent of RSL3’s presumed target, glutathione peroxidase 4. Investigating further, they found that RSL3 inhibits multiple antioxidant proteins in the peroxidase and selenoprotein families to more broadly affect reactive oxygen species and lipid peroxidation than previously assumed. Loss of ALKBH8, a tRNA methyltransferase responsible for modifying the selenocysteine-specific tRNA, broadly decreased selenoprotein activity and induced ferroptosis in colorectal cancer. This work identifies the selenoproteome as a therapeutic target in colorectal cancer via induction of reactive oxygen species, lipid peroxidation, and ferroptosis and adds to a growing body of literature on the potential utility of pro-oxidant mechanisms in cancer therapy. See related article by DeAngelo et al., p. 2788

Inflammasomes, particularly NLRC4, play a crucial role in immune responses to intracellular bacterial infections. However, gain-of-function mutations of NLRC4 are linked to severe autoinflammatory diseases, including autoinflammation with infantile enterocolitis (AIFEC). Existing mouse models do not adequately replicate the chronic, unprovoked inflammation observed in AIFEC patients. In this study, we developed a loxP-flanked NLRC4 V341A knock-in (KI) mouse model. Our global NLRC4 V341A KI mice exhibited symptoms closely mirroring those of human AIFEC. These mice demonstrated severe infantile enterocolitis, characterized by heightened intestinal inflammation, compromised intestinal barrier integrity, disrupted gut epithelium, and severe diarrhea, with high mortality within 10 days postnatally. Additionally, they displayed systemic autoinflammation marked by elevated levels of IL-1β, IL-18, and IL-6, alongside cytopenia and hemophagocytosis. In contrast, adult NLRC4 conditional KI mice exhibited autoinflammation with only mild enterocolitis. Since AIFEC is characterized by life-threatening enterocolitis in infancy and recurrent severe autoinflammation throughout life, our conditional NLRC4 KI model effectively recapitulates the clinical features of human AIFEC. Moreover, the NLRC4 V341A-mediated monogenic infantile enterocolitis observed in our model resembles infantile-onset inflammatory bowel disease (IBD), positioning it as a valuable platform for research on very early-onset IBD.

Undifferentiated intestinal stem cells (ISCs) are crucial for maintaining homeostasis and resolving injury. Lgr5+ cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis where they differentiate into specialized cell types. Coordinated execution of complex transcriptional programs is necessary to allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1^−/− mice, we have assessed the effects of MTGR1 loss specifically in ISC biology. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5, the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic and functional analyses revealed deficiencies in Mtgr1-null ISCs, including deregulated ISC-associated transcriptional programs. Ex vivo, intestinal organoids established from Mtgr1-null mice were unable to survive and expand due to aberrant differentiation and loss of stem and proliferative cells. Together, these results indicate that the role of MTGR1 in intestinal differentiation is likely stem cell intrinsic and identify a novel role for MTGR1 in maintaining ISC function.

Leucine-rich repeat kinase 2 (LRRK2) is a serine–threonine protein kinase belonging to the ROCO protein family. Within the kinase domain of LRRK2, a point mutation known as LRRK2 G2019S has emerged as the most prevalent variant associated with Parkinson’s disease. Recent clinical studies have indicated that G2019S carriers have an elevated risk of cancers, including colon cancer. Despite this observation, the underlying mechanisms linking LRRK2 G2019S to colon cancer remain elusive. In this study, employing a colitis-associated cancer (CAC) model and LRRK2 G2019S knock-in (KI) mouse model, we demonstrate that LRRK2 G2019S promotes the pathogenesis of colon cancer, characterized by increased tumor number and size in KI mice. Furthermore, LRRK2 G2019S enhances intestinal epithelial cell proliferation and inflammation within the tumor microenvironment. Mechanistically, KI mice exhibit heightened susceptibility to DSS-induced colitis, with inhibition of LRRK2 kinase activity ameliorating colitis severity and CAC progression. Our investigation also reveals that LRRK2 G2019S promotes inflammasome activation and exacerbates gut epithelium necrosis in the colitis model. Notably, GSDMD inhibitors attenuate colitis in LRRK2 G2019S KI mice. Taken together, our findings offer experimental evidence indicating that the gain-of-kinase activity in LRRK2 promotes colorectal tumorigenesis, suggesting LRRK2 as a potential therapeutic target in colon cancer patients exhibiting hyper LRRK2 kinase activity.

Introduction: The pro-inflammatory cytokine interleukin-23 (IL-23) has been implicated in colorectal cancer (CRC). Yet, the cell-specific contributions of IL-23 receptor (IL-23R) signaling in CRC remain unknown. One of the cell types that highly expresses IL-23R are colonic regulatory T cells (Treg cells). The aim of this study was to define the contribution of Treg cell-specific IL-23R signaling in sporadic and inflammation-associated CRC.Methods: In mice, the role of IL-23R in Treg cells in colitis-associated cancer (CAC) was investigated using azoxymethane/dextran sodium sulphate in wild-type Treg cell reporter mice (WT, Foxp3^YFP-iCre), and mice harboring a Treg cell-specific deletion of IL-23 (Il23r^ΔTreg). The role of IL-23R signaling in Treg cells in sporadic CRC was examined utilizing orthotopic injection of the syngeneic colon cancer cell line MC-38 submucosally into the colon/rectum of mice. The function of macrophages was studied using clodronate. Finally, single-cell RNA-seq of a previously published dataset in human sporadic cancer was reanalyzed to corroborate these findings.Results: In CAC, Il23r^ΔTreg mice had increased tumor size and increased dysplasia compared to WT mice that was associated with decreased tumor-infiltrating macrophages. In the sporadic cancer model, Il23r^ΔTreg mice had increased survival and decreased tumor size compared to WT mice. Additionally, MC-38 tumors of Il23r^ΔTreg mice exhibited a higher frequency of pro-inflammatory macrophages and IL-17 producing CD4⁺ T cells. The decreased tumor size in Il23r^ΔTreg mice was macrophage-dependent. These data suggest that loss of IL-23R signaling in Treg cells permits IL-17 production by CD4⁺ T cells that in turn promotes pro-inflammatory macrophages to clear tumors. Finally, analysis of TCGA data and single-cell RNA-seq analysis of a previously published dataset in human sporadic cancer, revealed that IL23R was highly expressed in CRC compared to other cancers and specifically in tumor-associated Treg cells.Conclusion: Inflammation in colorectal carcinogenesis differs with respect to the contribution of IL-23R signaling in regulatory T cells.

Background/Aims: Eosinophils are present in several solid tumors and have context-dependent function. Our aim is to define the contribution of eosinophils in esophageal squamous cell carcinoma (ESCC), since their role in ESCC is unknown. Methods: Eosinophils were enumerated in tissues from two ESCC cohorts. Mice were treated with 4-nitroquinolone-1-oxide (4-NQO) for 8 weeks to induce pre-cancer or 16 weeks to induce carcinoma. Eosinophil number was modified by monoclonal antibody to IL-5 (IL5mAb), recombinant IL-5 (rIL-5), or genetically with eosinophil-deficient (ΔdblGATA) mice or mice deficient in eosinophil chemoattractant eotaxin-1 ( Ccl11 ^-/- ). Esophageal tissue and eosinophil specific RNA-sequencing was performed to understand eosinophil function. 3-D co-culturing of eosinophils with pre-cancer or cancer cells was done to ascertain direct effects of eosinophils. Results: Activated eosinophils are present in higher numbers in early stage versus late stage ESCC. Mice treated with 4-NQO exhibit more esophageal eosinophils in pre-cancer versus cancer. Correspondingly, epithelial cell Ccl11 expression is higher in mice with pre-cancer. Eosinophil depletion using three mouse models ( Ccl11 ^-/- mice, ΔdblGATA mice, IL5mAb treatment) all display exacerbated 4-NQO tumorigenesis. Conversely, treatment with rIL-5 increases esophageal eosinophilia and protects against pre-cancer and carcinoma. Tissue and eosinophil RNA-sequencing revealed eosinophils drive oxidative stress in pre-cancer. In vitro co-culturing of eosinophils with pre-cancer or cancer cells resulted in increased apoptosis in the presence of a degranulating agent, which is reversed with N-acetylcysteine, a reactive oxygen species (ROS) scavenger. ΔdblGATA mice exhibited increased CD4 T cell infiltration, IL-17, and enrichment of IL-17 pro-tumorigenic pathways. Conclusion: Eosinophils likely protect against ESCC through ROS release during degranulation and suppression of IL-17.

Supplemental Figure 1. STK17A expression does not correlate with CRC patient survival. Supplemental Figure 2. Attenuated morphological characteristics observed with decreased STK17A KD. Supplemental Figure 3. STK17A does not affect proliferation or viability of CRC cells. Supplemental Figure 4. STK17A expression KD human tumor organoid lines. Supplemental Figure 5. No phenotype induced by STK17A loss in HTO3.

Supplemental Figure 1. STK17A expression does not correlate with CRC patient survival. Supplemental Figure 2. Attenuated morphological characteristics observed with decreased STK17A KD. Supplemental Figure 3. STK17A does not affect proliferation or viability of CRC cells. Supplemental Figure 4. STK17A expression KD human tumor organoid lines. Supplemental Figure 5. No phenotype induced by STK17A loss in HTO3.

Supplemental Table 1. qRT-PCR and mutagenesis primer sequences. Supplemental Table 2. Clinical features of human tumors from which organoid lines were derived. Supplemental Table 3. Components of complete human organoid media.

Serine threonine kinase 17A (STK17A) is a ubiquitously expressed kinase originally identified as a regulator of apoptosis; however, whether it functionally contributes to colorectal cancer has not been established. Here, we have analyzed STK17A in colorectal cancer and demonstrated decreased expression of STK17A in primary tumors, which is further reduced in metastatic lesions, indicating a potential role in regulating the metastatic cascade. Interestingly, changes in STK17A expression did not modify proliferation, apoptosis, or sensitivity of colorectal cancer cell lines to treatment with the chemotherapeutic 5-fluorouracil. Instead, STK17A knockdown induced a robust mesenchymal phenotype consistent with the epithelial–mesenchymal transition, including spindle-like cell morphology, decreased expression of adherens junction proteins, and increased migration and invasion. Additionally, overexpression of STK17A decreased cell size and induced widespread membrane blebbing, a phenotype often associated with activation of cell contractility. Indeed, STK17A-overexpressing cells displayed heightened phosphorylation of myosin light chain in a manner dependent on STK17A catalytic activity. Finally, patient-derived tumor organoid cultures were used to more accurately determine STK17A's effect in primary human tumor cells. Loss of STK17A induced morphologic changes, decreased E-cadherin, increased invasion, and augmented organoid attachment on 2D substrates, all together suggesting a more metastatic phenotype. Collectively, these data indicate a novel role for STK17A in the regulation of epithelial phenotypes and indicate its functional contribution to colorectal cancer invasion and metastasis.Implications:Loss of serine threonine kinase 17A occurs in colorectal cancer metastasis, induces mesenchymal morphologies, and contributes to tumor cell invasion and migration in colorectal cancer.