Annalisa L. E. Carli

Doublecortin-like kinase 1 (DCLK1) is a proposed driver of gastric cancer (GC) that phosphorylates serine and threonine residues. Here, we showed that the kinase activity of DCLK1 orchestrated cancer cell–intrinsic and–extrinsic processes that led to pro-invasive and pro-metastatic reprogramming of GC cells. Inhibition of the kinase activity of DCLK1 reduced the growth of subcutaneous xenograft tumors formed from MKN1 human gastric carcinoma cells in mice and decreased the abundance of the stromal markers α-Sma, vimentin, and collagen. Similar effects were seen in mice with xenograft tumors formed from MKN1 cells expressing a kinase-inactive DCLK1 mutant (MKN1 ^D511N ). MKN1 ^D511N cells also had reduced in vitro migratory potential and stemness compared with control cells. Mice orthotopically grafted with MKN1 cells overexpressing DCLK1 (MKN1 ^DCLK1 ) showed increased invasiveness and had a greater incidence of lung metastases compared with those grafted with control MKN1 cells. Mechanistically, we showed that the chemokine CXCL12 acted downstream of DCLK1 in cultured MKN1 cells and in mice subcutaneously implanted with gastric tumors formed by MKN1 ^DCLK1 cells. Moreover, inhibition of the kinase activity of DCLK1 or the expression of DCLK1 ^D511N reversed the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a broadly acting and potentially targetable promoter of GC.

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest a role for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing an opportunity to therapeutically inhibit early-stage gastric cancer through repurposing antibody-mediated therapies.

Specific signalling thresholds of the WNT/β-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/β-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/β-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene APC that otherwise prevents prolonged stabilisation of β-catenin. Surprisingly, no inhibitor compounds for WNT/β-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal Apc^flox/flox mice can rescue in utero death of Apc^min/flox mutant conceptus without subsequent post-mortem assessment of WNT/β-catenin signalling. Indeed, the phenotypic plasticity of born Apc^min/flox conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/β-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/β-catenin antagonists.

Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αβ-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.

Gastric cancer (GC) is the 3rd leading cause of cancer mortality worldwide, therefore providing novel diagnostic and treatment options is crucial for at risk groups. The serine/threonine kinase doublecortin-like kinase 1 (DCLK1) is a proposed driver of GC with frequent amplification and somatic missense mutations yet the molecular mechanism how DCLK1 mediates tumorigenesis is poorly understood. We report how DCLK1 expression orchestrates complementary cancer cell intrinsic and extrinsic processes leading to a comprehensive pro-invasive and pro-metastatic reprogramming of cancer cells and tumor stroma in a DCLK1 kinase-dependent manner. Mechanistically, we identify the chemokine CXCL12 as a key promoter of the pro-tumorigenic properties downstream of DCLK1. Importantly, inhibition of the DCLK1 kinase domain reverses the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a promising, targetable master regulator of GC. Teaser DCLK1 is a druggable cancer driver of GC

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here we explore a metabolite-triggered circuit between epithelial tuft cells and innate lymphoid type 2 cells (ILC2) that is evolutionarily optimized for intestinal remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) acts as an alarmin on ILC2s to induce the release of IL13 as a growth factor for tuft cells, and propose that this model drives early metaplastic remodeling and gastric tumor formation. Genetic ablation of tuft cells, ILC2s or antibody-mediated neutralization of IL13 or IL25 reduces the growth of established tumors. Thus, the tuft cell/ILC2 axis provides an opportunity to therapeutically inhibit preneoplastic lesions and early-stage gastric cancer through repurposing of antibody-mediated therapies. One-Sentence Summary Tuft cells and type 2 innate lymphoid cells offer a new therapeutic target in gastric disease.

MicroRNA-21 (miR-21) is a small, non-coding RNA overexpressed in gastric cancer and many other solid malignancies, where it exhibits both pro-and anti-tumourigenic properties. However, the pathways regulating miR-21 and the consequences of its inhibition in gastric cancer remain incompletely understood. By exploiting the spontaneous Stat3-dependent formation of inflammation-associated gastric tumors in Gp130^F/F mice, we functionally established miR-21 as a Stat3-controlled driver of tumor growth and progression. We reconciled our discoveries by identifying several conserved Stat3 binding motifs upstream of the miR-21 gene promoter, and showed that the systemic administration of a miR-21-specific antisense oligonucleotide antagomir reduced the established gastric tumor burden in Gp130^F/F mice. We molecularly delineated the therapeutic benefits of miR-21 inhibition with the functional restoration of PTEN in vitro and in vivo, alongside an attenuated epithelial-to-mesenchymal transition and the extracellular matrix remodeling phenotype of tumors. We corroborated our preclinical findings by correlating high STAT3 and miR-21 expression with the reduced survival probability of gastric cancer patients. Collectively, our results provide a molecular framework by which miR-21 mediates inflammation-associated gastric cancer progression, and establish miR-21 as a robust therapeutic target for solid malignancies characterized by excessive Stat3 activity.

Doublecortin‐like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor‐prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial‐to‐mesenchymal transition (EMT), driving disruption of cell‐cell adhesion, cell migration and invasion. Here, we report that DCLK1 influences small extracellular vesicle (sEV/exosome) biogenesis in a kinase‐dependent manner. sEVs isolated from DCLK1 overexpressing human GC cell line MKN1 (MKN1^OE‐sEVs), promote the migration of parental (non‐transfected) MKN1 cells (MKN1^PAR). Quantitative proteome analysis of MKN1^OE‐sEVs revealed enrichment in migratory and adhesion regulators (STRAP, CORO1B, BCAM, COL3A, CCN1) in comparison to MKN1^PAR‐sEVs. Moreover, using DCLK1‐IN‐1, a specific small molecule inhibitor of DCLK1, we reversed the increase in sEV size and concentration in contrast to other EV subtypes, as well as kinase‐dependent cargo selection of proteins involved in EV biogenesis (KTN1, CHMP1A, MYO1G) and migration and adhesion processes (STRAP, CCN1). Our findings highlight a specific role of DCLK1‐kinase dependent cargo selection for sEVs and shed new light on its role as a regulator of signaling in gastric tumorigenesis.