José Manuel González-Sancho

Early-onset colorectal cancer (EO-CRC, <50 years) incidence is rising worldwide. Using paired patient-derived colorectal normal and tumor organoids, we demonstrate the therapeutic window of three standard chemotherapies. Normal organoids were more resistant than tumor organoids to 5-fluorouracil (5-FU) and oxaliplatin in both EO-CRC and late-onset CRC (LO-CRC) patients. However, for SN38, this therapeutic window was observed in LO-CRC but not in EO-CRC patients, revealing age-dependent differences in drug resistance. We also evaluated the effect of calcitriol, the active vitamin D metabolite with anti-CRC activity, on drug sensitivity. Calcitriol reduced the cytotoxicity of 5-FU and SN38 in normal organoids regardless of patient age, while it selectively decreased their cytotoxicity in EO-CRC but not in LO-CRC tumor organoids. This protective effect correlated with calcitriol antiproliferative action and transcriptional effects on drug-metabolism pathways. These findings identify clinically relevant age-dependent differences in chemotherapy response and support vitamin D-based strategies to design age-tailored precision treatments.

Cadherin 17 (CDH17) plays a crucial role in the metastatic progression of colorectal cancer (CRC) through its interaction with α2β1 integrin and desmocollin 1. To further elucidate the molecular mechanisms involving CDH17 functions in CRC, we examined global expression alterations following CDH17 silencing in various metastatic cell lines. Loss of CDH17 resulted in a marked down-regulation of the intestinal cancer stem cell (CSC) marker LGR5, leading to the inhibition of Wnt/β-catenin signaling, suppression of pluripotency genes such as MYC, and a subsequent reduction in stemness properties. Treatment with CDH17/integrin blocking antibodies produced similar effects, decreasing both, LGR5 expression and Wnt signaling. CDH17 silencing also down-regulated various transporters associated with drug-resistance, including the glutamine-transporter SLC38A5. Consequently, the loss of CDH17 increased sensitivity to 5-FU, irinotecan, oxidative stress and anoikis in CRC cells. Notably, SLC38A5 silencing was necessary for CDH17-driven effects on drug resistance and survival. Pharmacological inhibition of SLC38A5 with amiloride, significantly increased cell sensitivity to 5-FU and irinotecan, and improved mouse survival in metastasis models. In conclusion, CDH17 plays a crucial role in maintaining colorectal cancer cell stemness and chemoresistance via LGR5/Wnt/MYC signaling and SLC38A5 expression. These findings underscore the therapeutic potential of CDH17 targeting in metastatic CRC, and support the use of amiloride for inhibiting liver metastasis.

Cadherin 17 (CDH17) plays a crucial role in the metastatic progression of colorectal cancer (CRC) through its interaction with α2β1 integrin. To further elucidate the molecular mechanisms involving CDH17 functions in CRC, we examined global expression alterations following CDH17 silencing in various metastatic cell lines. Loss of CDH17 resulted in a marked down-regulation of the cancer stem cell (CSC) marker LGR5, leading to the inhibition of Wnt/β-catenin signaling, suppression of pluripotency genes like MYC, and a subsequent reduction in stemness properties. Mechanistically, treatment with CDH17-blocking antibodies produced similar effects, decreasing both, LGR5 expression and Wnt signaling. CDH17 silencing also down-regulated various transporters associated with drug-resistance, including the glutamine-transporter SLC38A5, thereby enhancing sensitivity to 5-FU, irinotecan, oxidative stress and anoikis. Silencing of SLC38A5 via siRNA further amplified the effects of CDH17 on drug sensitivity, oxidative stress, and anoikis. Furthermore, treatment with amiloride, an SLC38A5 inhibitor, significantly reduced cell viability in response to 5-FU and irinotecan, and improved survival in mouse metastasis models. In conclusion, CDH17 expression is critical for maintaining the signaling cascade Wnt/MYC, responsible of the CSC traits, and drug resistance properties (mediated by SLC38A5) in CRC. These findings underscore the therapeutic potential of targeting CDH17 in metastatic CRC and support the use of amiloride as a promising strategy for inhibiting liver metastasis.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D₃ (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Colorectal cancer (CRC) is one of the most life‐threatening neoplasias in terms of incidence and mortality worldwide. Vitamin D deficiency has been associated with an increased risk of CRC. 1α,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃], the most active vitamin D metabolite, is a pleiotropic hormone that, through its binding to a transcription factor of the nuclear receptor superfamily, is a major regulator of the human genome. 1,25(OH)₂D₃ acts on colon carcinoma and stromal cells and displays tumor protective actions. Here, we review the variety of molecular mechanisms underlying the effects of 1,25(OH)₂D₃ in CRC, which affect multiple processes that are dysregulated during tumor initiation and progression. Additionally, we discuss the epidemiological data that associate vitamin D deficiency and CRC, and the most relevant randomized controlled trials of vitamin D₃ supplementation conducted in both healthy individuals and CRC patients.

The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)₂D₃ and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)₂D₃ and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)₂D₃ and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)₂D₃ reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)₂D₃ and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/β‐catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD‐18Co cells. A set of genes encoding inhibitors of the Wnt/β‐catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin‐2 is a novel direct transcriptional target of Wnt/β‐catenin in normal and colon cancer‐associated fibroblasts. PKP2 is induced by β‐catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin‐2 antagonizes Wnt/β‐catenin transcriptional activity in HEK‐293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β‐catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin‐2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

SPROUTY-2 (SPRY2) is a modulator of tyrosine kinase receptor signaling with receptor- and cell type-dependent inhibitory or enhancing effects. Studies on the action of SPRY2 in major cancers are conflicting and its role remains unclear. Here we have dissected SPRY2 action in human colon cancer. Global transcriptomic analyses show that SPRY2 downregulates genes encoding tight junction proteins such as claudin-7 and occludin and other cell-to-cell and cell-to-matrix adhesion molecules in human SW480-ADH colon carcinoma cells. Moreover, SPRY2 represses LLGL2/HUGL2, PATJ1/INADL and ST14, main regulators of the polarized epithelial phenotype, and ESRP1, an epithelial-to-mesenchymal transition (EMT) inhibitor. A key action of SPRY2 is the upregulation of the major EMT inducer ZEB1, as these effects are reversed by ZEB1 knock-down by means of RNA interference. Consistently, we found an inverse correlation between the expression level of claudin-7 and those of SPRY2 and ZEB1 in human colon tumors. Mechanistically, ZEB1 upregulation by SPRY2 results from the combined induction of ETS1 transcription factor and the repression of microRNAs (miR-200 family, miR-150) that target ZEB1 RNA. Moreover, SPRY2 increased AKT activation by epidermal growth factor, whereas AKT and also Src inhibition reduced the induction of ZEB1. Altogether, these data suggest that AKT and Src are implicated in SPRY2 action. Collectively, these results show a tumorigenic role of SPRY2 in colon cancer that is based on the dysregulation of tight junction and epithelial polarity master genes via upregulation of ZEB1. The dissection of the mechanism of action of SPRY2 in colon cancer cells is important to understand the upregulation of this gene in a subset of patients with this neoplasia that have poor prognosis.

Aims: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. Results: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. Innovation: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. Conclusion: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism. Antioxid. Redox Signal. 22, 555–571.