Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

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.

Clinico-Pathological Features, Outcomes and Impacts of COVID-19 Pandemic on Patients with Early-Onset Colorectal Cancer: A Single-Institution Experience.

BACKGROUND: The rising incidence of colorectal cancer (CRC) among young patients is alarming. We aim to characterize the clinico-pathological features and outcomes of patients with early-onset CRC (EOCRC), as well as the impacts of COVID-19 pandemic. METHODS: We included all patients with pathologically confirmed diagnoses of CRC at Hospital Universitario La Paz from October 2016 to December 2021. The EOCRC cut-off age was 50 years old. RESULTS: A total of 1475 patients diagnosed with CRC were included, eighty (5.4%) of whom had EOCRC. Significant differences were found between EOCRC and later-onset patients regarding T, N stage and metastatic presentation at diagnosis; perineural invasion; tumor budding; high-grade tumors; and signet ring cell histology, with all issues having higher prevalence in the early-onset group. More EOCRC patients had the RAS/ BRAF wild type. Chemotherapy was administered more frequently to patients with EOCRC. In the metastatic setting, the EOCRC group presented a significantly longer median OS. Regarding the COVID-19 pandemic, more patients with COVID-19 were diagnosed with metastatic disease (61%) in the year after the lockdown (14 March 2020) than in the pre-pandemic EOCRC group (29%). CONCLUSIONS: EOCRC is diagnosed at a more advanced stage and with worse survival features in localized patients. More patients with EOCRC were diagnosed with metastatic disease in the year after the COVID-19 pandemic lockdown. The long-term consequences of COVID-19 are yet to be determined.

CDK5RAP3, a New BRCA2 Partner That Regulates DNA Repair, Is Associated with Breast Cancer Survival.

BRCA2 is essential for homologous recombination DNA repair. BRCA2 mutations lead to genome instability and increased risk of breast and ovarian cancer. Similarly, mutations in BRCA2-interacting proteins are also known to modulate sensitivity to DNA damage agents and are established cancer risk factors. Here we identify the tumor suppressor CDK5RAP3 as a novel BRCA2 helical domain-interacting protein. CDK5RAP3 depletion induced DNA damage resistance, homologous recombination and single-strand annealing upregulation, and reduced spontaneous and DNA damage-induced genomic instability, suggesting that CDK5RAP3 negatively regulates double-strand break repair in the S-phase. Consistent with this cellular phenotype, analysis of transcriptomic data revealed an association between low CDK5RAP3 tumor expression and poor survival of breast cancer patients. Finally, we identified common genetic variations in the CDK5RAP3 locus as potentially associated with breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Our results uncover CDK5RAP3 as a critical player in DNA repair and breast cancer outcomes.

Organoids and Colorectal Cancer.

Organoids were first established as a three-dimensional cell culture system from mouse small intestine. Subsequent development has made organoids a key system to study many human physiological and pathological processes that affect a variety of tissues and organs. In particular, organoids are becoming very useful tools to dissect colorectal cancer (CRC) by allowing the circumvention of classical problems and limitations, such as the impossibility of long-term culture of normal intestinal epithelial cells and the lack of good animal models for CRC. In this review, we describe the features and current knowledge of intestinal organoids and how they are largely contributing to our better understanding of intestinal cell biology and CRC genetics. Moreover, recent data show that organoids are appropriate systems for antitumoral drug testing and for the personalized treatment of CRC patients.

Comparative Study of Organoids from Patient-Derived Normal and Tumor Colon and Rectal Tissue.

Colon and rectal tumors, often referred to as colorectal cancer, show different gene expression patterns in studies that analyze whole tissue biopsies containing a mix of tumor and non-tumor cells. To better characterize colon and rectal tumors, we investigated the gene expression profile of organoids generated from endoscopic biopsies of rectal tumors and adjacent normal colon and rectum mucosa from therapy-naive rectal cancer patients. We also studied the effect of vitamin D on these organoid types. Gene profiling was performed by RNA-sequencing. Organoids from a normal colon and rectum had a shared gene expression profile that profoundly differed from that of rectal tumor organoids. We identified a group of genes of the biosynthetic machinery as rectal tumor organoid-specific, including those encoding the RNA polymerase II subunits POLR2H and POLR2J. The active vitamin D metabolite 1α,25-dihydroxyvitamin D3/calcitriol upregulated stemness-related genes (LGR5, LRIG1, SMOC2, and MSI1) in normal rectum organoids, while it downregulated differentiation marker genes (TFF2 and MUC2). Normal colon and rectum organoids share similar gene expression patterns and respond similarly to calcitriol. Rectal tumor organoids display distinct and heterogeneous gene expression profiles, with differences with respect to those of colon tumor organoids, and respond differently to calcitriol than normal rectum organoids.

Vitamin D Effects on Cell Differentiation and Stemness in Cancer.

Vitamin D3 is the precursor of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), a pleiotropic hormone that is a major regulator of the human genome. 1,25(OH)2D3 modulates the phenotype and physiology of many cell types by controlling the expression of hundreds of genes in a tissue- and cell-specific fashion. Vitamin D deficiency is common among cancer patients and numerous studies have reported that 1,25(OH)2D3 promotes the differentiation of a wide panel of cultured carcinoma cells, frequently associated with a reduction in cell proliferation and survival. A major mechanism of this action is inhibition of the epithelial-mesenchymal transition, which in turn is largely based on antagonism of the Wnt/ß-catenin, TGF-ß and EGF signaling pathways. In addition, 1,25(OH)2D3 controls the gene expression profile and phenotype of cancer-associated fibroblasts (CAFs), which are important players in the tumorigenic process. Moreover, recent data suggest a regulatory role of 1,25(OH)2D3 in the biology of normal and cancer stem cells (CSCs). Here, we revise the current knowledge of the molecular and genetic basis of the regulation by 1,25(OH)2D3 of the differentiation and stemness of human carcinoma cells, CAFs and CSCs. These effects support a homeostatic non-cytotoxic anticancer action of 1,25(OH)2D3 based on reprogramming of the phenotype of several cell types.

Vitamin D differentially regulates colon stem cells in patient-derived normal and tumor organoids.

Intestine is a major target of vitamin D and several studies indicate an association between vitamin D deficiency and inflammatory bowel diseases (IBD), but also increased colorectal cancer (CRC) risk and mortality. However, the putative effects of 1α,25-dihydroxyvitamin D3 (calcitriol), the active vitamin D metabolite, on human colonic stem cells are unknown. Here we show by immunohistochemistry and RNAscope in situ hybridization that vitamin D receptor (VDR) is unexpectedly expressed in LGR5+ colon stem cells in human tissue and in normal and tumor organoid cultures generated from patient biopsies. Interestingly, normal and tumor organoids respond differentially to calcitriol with profound and contrasting changes in their transcriptomic profiles. In normal organoids, calcitriol upregulates stemness-related genes, such as LGR5, SMOC2, LRIG1, MSI1, PTK7, and MEX3A, and inhibits cell proliferation. In contrast, in tumor organoids calcitriol has little effect on stemness-related genes while it induces a differentiated phenotype, and variably reduces cell proliferation. Concordantly, electron microscopy showed that calcitriol does not affect the blastic undifferentiated cell phenotype in normal organoids but it induces a series of differentiated features in tumor organoids. Our results constitute the first demonstration of a regulatory role of vitamin D on human colon stem cells, indicating a homeostatic effect on colon epithelium with relevant implications in IBD and CRC.

Plocabulin Displays Strong Cytotoxic Activity in a Personalized Colon Cancer Patient-Derived 3D Organoid Assay.

Plocabulin is a novel microtubule-disrupting antitumor agent of marine origin that is currently undergoing phase II clinical trials. Plocabulin has potent antiproliferative and antiangiogenic actions in carcinoma cell lines and has antitumor activity in xenografted mice. Here, we used three-dimensional (3D) tumor organoids derived from three colorectal cancer (CRC) patients to study the effect of plocabulin in a personalized assay system that ensures dose dependence and high reproducibility. The cytotoxicity of plocabulin was an order of magnitude higher than that of the active irinotecan derivative SN38 (7-ethyl-10-hydroxy-camptothecin) in tumor organoids at different passages. Moreover, plocabulin maintained its strong cytotoxic activity in wash-out experiments, in which a short pulse treatment of tumor organoids was as efficient as continuous treatment. Our data show that plocabulin has a very potent cytotoxic action in CRC patient-derived tumor organoids, supporting ongoing clinical trials with plocabulin and the use of organoid assays to provide personalized validation of antitumor drugs.