Myoferlin: A Potential Marker of Response to Radiation Therapy and Survival in Locally Advanced Rectal Cancer.

PURPOSE: Patients with locally advanced rectal cancer often require neoadjuvant chemoradiation therapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiation therapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target. METHODS AND MATERIALS: Immunohistochemical analysis of a tissue microarray (TMA) for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using small interfering RNA, a small molecular inhibitor (wj460), and a CRISPR-Cas9 knockout cell line. Radiosensitization after treatment was assessed using 2-dimensional clonogenic assays, 3-dimensional spheroid models, and patient-derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence, and immunoblotting. RESULTS: Analysis of both the diagnostic biopsy and tumor resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant long-course chemoradiation therapy. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (P = .01; hazard ratio, 3.5; 95% CI, 1.27-10.04). This was externally validated using the Stratification in Colorectal Cancer database. Quantification of myoferlin using immunoblotting in immortalized colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacologic manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2-dimensional and 3-dimensional models. After irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double-strand breaks. This appeared to be mediated via nonhomologous end-joining. CONCLUSIONS: We have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of patients with rectal cancer to radiation therapy, and further work is required.

CARE: Protocol of a randomised trial evaluating the feasibility of preoperative intentional weight loss to support postoperative recovery in patients with excess weight and colorectal cancer.

AIM: Excess weight increases the risk of morbidity following colorectal cancer surgery. Weight loss may improve morbidity, but it is uncertain whether patients can follow an intensive weight loss intervention while waiting for surgery and there are concerns about muscle mass loss. The aim of this trial is to assess the feasibility of intentional weight loss in this setting and determine progression to a definitive trial. METHODS: CARE is a prospectively registered, multicentre, feasibility, parallel, randomised controlled trial with embedded evaluation and optimisation of the recruitment process. Participants with excess weight awaiting curative colorectal resection for cancer are randomised 1:1 to care as usual or a low-energy nutritionally-replete total diet replacement programme with weekly remote behavioural support by a dietitian. Progression criteria will be based on the recruitment, engagement, adherence, and retention rates. Data will be collected on the 30-day postoperative morbidity, the typical primary outcome of prehabilitation trials. Secondary outcomes will include, among others, length of hospital stay, health-related quality of life, and body composition. Qualitative interviews will be used to understand patients' experiences of and attitudes towards trial participation and intervention engagement and adherence. CONCLUSION: CARE will evaluate the feasibility of intensive intentional weight loss as prehabilitation before colorectal cancer surgery. The results will determine the planning of a definitive trial.

Inherited MUTYH mutations cause elevated somatic mutation rates and distinctive mutational signatures in normal human cells.

Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequence normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 4-fold in all individuals, except for one showing a 31-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C > A changes. Different mutation rates and signatures between individuals are likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.

An innate IL-25-ILC2-MDSC axis creates a cancer-permissive microenvironment for Apc mutation-driven intestinal tumorigenesis.

Interleukin-25 (IL-25) and group 2 innate lymphoid cells (ILC2s) defend the host against intestinal helminth infection and are associated with inappropriate allergic reactions. IL-33-activated ILC2s were previously found to augment protective tissue-specific pancreatic cancer immunity. Here, we showed that intestinal IL-25-activated ILC2s created an innate cancer-permissive microenvironment. Colorectal cancer (CRC) patients with higher tumor IL25 expression had reduced survival and increased IL-25R-expressing tumor-resident ILC2s and myeloid-derived suppressor cells (MDSCs) associated with impaired antitumor responses. Ablation of IL-25 signaling reduced tumors, virtually doubling life expectancy in an Apc mutation-driven model of spontaneous intestinal tumorigenesis. Mechanistically, IL-25 promoted intratumoral ILC2s, which sustained tumor-infiltrating MDSCs to suppress antitumor immunity. Therapeutic antibody-mediated blockade of IL-25 signaling decreased intratumoral ILC2s, MDSCs, and adenoma/adenocarcinoma while increasing antitumor adaptive T cell and interferon-γ (IFN-γ)-mediated immunity. Thus, the roles of innate epithelium-derived cytokines IL-25 and IL-33 as well as ILC2s in cancer cannot be generalized. The protumoral nature of the IL-25-ILC2 axis in CRC highlights this pathway as a potential therapeutic target against CRC.

Mutational landscape of normal epithelial cells in Lynch Syndrome patients.

Lynch Syndrome (LS) is an autosomal dominant disease conferring a high risk of colorectal cancer due to germline heterozygous mutations in a DNA mismatch repair (MMR) gene. Although cancers in LS patients show elevated somatic mutation burdens, information on mutation rates in normal tissues and understanding of the trajectory from normal to cancer cell is limited. Here we whole genome sequence 152 crypts from normal and neoplastic epithelial tissues from 10 LS patients. In normal tissues the repertoire of mutational processes and mutation rates is similar to that found in wild type individuals. A morphologically normal colonic crypt with an increased mutation burden and MMR deficiency-associated mutational signatures is identified, which may represent a very early stage of LS pathogenesis. Phylogenetic trees of tumour crypts indicate that the most recent ancestor cell of each tumour is already MMR deficient and has experienced multiple cycles of clonal evolution. This study demonstrates the genomic stability of epithelial cells with heterozygous germline MMR gene mutations and highlights important differences in the pathogenesis of LS from other colorectal cancer predisposition syndromes.

5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation.

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Itraconazole perturbs colorectal cancer dormancy through SUFU-mediated WNT inhibition.

Cancer cell dormancy is an important source of treatment failure. We studied the molecular characteristics and functional behaviour of dormant colorectal cancer cells finding them to be a differentiated yet plastic population. Organoid drug screening identified itraconazole perturbs dormancy through non-canonical hedgehog signalling effects on the WNT pathway.

Itraconazole targets cell cycle heterogeneity in colorectal cancer.

Cellular dormancy and heterogeneity in cell cycle length provide important explanations for treatment failure after adjuvant therapy with S-phase cytotoxics in colorectal cancer (CRC), yet the molecular control of the dormant versus cycling state remains unknown. We sought to understand the molecular features of dormant CRC cells to facilitate rationale identification of compounds to target both dormant and cycling tumor cells. Unexpectedly, we demonstrate that dormant CRC cells are differentiated, yet retain clonogenic capacity. Mouse organoid drug screening identifies that itraconazole generates spheroid collapse and loss of dormancy. Human CRC cell dormancy and tumor growth can also be perturbed by itraconazole, which is found to inhibit Wnt signaling through noncanonical hedgehog signaling. Preclinical validation shows itraconazole to be effective in multiple assays through Wnt inhibition, causing both cycling and dormant cells to switch to global senescence. These data provide preclinical evidence to support an early phase trial of itraconazole in CRC.

Multipotent Basal Stem Cells, Maintained in Localized Proximal Niches, Support Directed Long-Ranging Epithelial Flows in Human Prostates.

Sporadic mitochondrial DNA mutations serve as clonal marks providing access to the identity and lineage potential of stem cells within human tissues. By combining quantitative clonal mapping with 3D reconstruction of adult human prostates, we show that multipotent basal stem cells, confined to discrete niches in juxta-urethral ducts, generate bipotent basal progenitors in directed epithelial migration streams. Basal progenitors are then dispersed throughout the entire glandular network, dividing and differentiating to replenish the loss of apoptotic luminal cells. Rare lineage-restricted luminal stem cells, and their progeny, are confined to proximal ducts and provide only minor contribution to epithelial homeostasis. In situ cell capture from clonal maps identified delta homolog 1 (DLK1) enrichment of basal stem cells, which was validated in functional spheroid assays. This study establishes significant insights into niche organization and function of prostate stem and progenitor cells, with implications for disease.

Mex3a Marks a Slowly Dividing Subpopulation of Lgr5+ Intestinal Stem Cells.

Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs).

Mouse models of colorectal cancer as preclinical models.

In this review, we discuss the application of mouse models to the identification and pre-clinical validation of novel therapeutic targets in colorectal cancer, and to the search for early disease biomarkers. Large-scale genomic, transcriptomic and epigenomic profiling of colorectal carcinomas has led to the identification of many candidate genes whose direct contribution to tumourigenesis is yet to be defined; we discuss the utility of cross-species comparative 'omics-based approaches to this problem. We highlight recent progress in modelling late-stage disease using mice, and discuss ways in which mouse models could better recapitulate the complexity of human cancers to tackle the problem of therapeutic resistance and recurrence after surgical resection.

Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells.

We report here that side population (SP) sorting allows for the simultaneous isolation of two intestinal stem cell (ISC) subsets from wild-type (WT) mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2'-deoxyuridine (EdU) injection, in the upper side population (USP) the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP), only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFP(hi) cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs) in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.

Colon resection: is standard technique adequate?

The traditional approach to surgical resection of colonic cancer involves removal of the primary tumor together with the associated lymphovascular pedicle. In an attempt to improve oncological outcomes, several groups have recently published data describing improved outcomes with a more radical surgical approach termed complete mesocolic excision (CME) with central vessel ligation (CVL). Here we critically appraise this new surgical advance and discuss other surgical options suggested to offer improvements over current best practice.

Intestinal label-retaining cells are secretory precursors expressing Lgr5.

The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.