SummaryDespite evidence for the impact of insulin on intestinal epithelial physiology and pathophysiology, the expression patterns, roles, and regulation of insulin receptor (IR) and IR isoforms in the intestinal epithelium are not well characterized. IR-A is thought to mediate the proliferative effects of insulin or insulin growth factors (IGFs) in fetal or cancer cells. IR-B is considered to be the metabolic receptor for insulin in specialized tissues. This study used a novel Sox9-EGFP reporter mouse that permits isolation of intestinal epithelial stem cells (IESCs), progenitors, enteroendocrine cells and differentiated lineages, the ApcMin/+ mouse model of precancerous adenoma and normal human intestinal and colorectal cancer (CRC) cell lines. We tested the hypothesis that there is differential expression of IR-A or IR-B in stem and tumor cells versus differentiated intestinal epithelial cells (IECs) and that IR-B impacts cell proliferation. Our findings provide evidence that IR-B expression is significantly lower in highly proliferative IESCs and progenitor cells versus post-mitotic, differentiated IECs and in subconfluent and undifferentiated versus differentiated Caco-2 cells. IR-B is also reduced in Apc Min/+ tumors and highly tumorigenic CRC cells. These differences in IR-B were accompanied by altered levels of mRNAs encoding muscleblind-like 2 (MBNL2), a known regulator of IR alternative splicing. Forced IR-B expression in subconfluent and undifferentiated Caco-2 cells reduced proliferation and increased biomarkers of differentiation. Our findings indicate that the impact of insulin on different cell types in the intestinal epithelium might differ depending on relative IR-B: IR-A expression levels and provide new evidence for the roles of IR-B to limit proliferation of CRC cells.

Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFPLow), activatable reserve IESC and enteroendocrine cells (Sox9-EGFPHigh), Sox9-EGFPSublow progenitors, and Sox9-EGFPNegative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFPLow IESC and Sox9-EGFPHigh cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.

The colon tumor microenvironment (TME) is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1LoxP/LoxP, or Imp1ΔMes) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histological grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in pro-tumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1ΔMes mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states.

The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IR(Δ/Δ)) and littermate controls with floxed, but intact, IR (IR(fl/fl)) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IR(Δ/Δ) mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IR(fl/fl) and VC-IR(Δ/Δ) mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IR(fl/fl) mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IR(Δ/Δ) mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs.

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified as a principle node for gene expression regulation downstream from In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion (Imp1 DIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1 DIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1 DIEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B, ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1 DIEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.

The RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) is overexpressed in colorectal cancer (CRC); however, evidence for a direct role for IMP1 in CRC metastasis is lacking. IMP1 is regulated by let-7 microRNA, which binds in the 3′ untranslated region (UTR) of the transcript. The availability of binding sites is in part controlled by alternative polyadenylation, which determines 3′ UTR length. Expression of the short 3′ UTR transcript (lacking all microRNA sites) results in higher protein levels and is correlated with increased proliferation. We used in vitro and in vivo model systems to test the hypothesis that the short 3′ UTR isoform of IMP1 promotes CRC metastasis. Herein we demonstrate that 3′ UTR shortening increases IMP1 protein expression and that this in turn enhances the metastatic burden to the liver, whereas expression of the long isoform (full length 3′ UTR) does not. Increased tumor burden results from elevated tumor surface area driven by cell proliferation and cell survival mechanisms. These processes are independent of classical apoptosis pathways. Moreover, we demonstrate the shifts toward the short isoform are associated with metastasis in patient populations where IMP1-long expression predominates. Overall, our work demonstrates that different IMP1 expression levels result in different functional outcomes in CRC metastasis and that targeting IMP1 may reduce tumor progression in some patients.

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform-expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a-dependent manner. The LIN28B-short isoform preserved its ability to bind pre-, without inhibiting the maturation of , and competed with the LIN28B-long isoform for binding to pre- Coexpression of the short isoform in the LIN28B-long isoform-expressing cells rescued the phenotypes induced by the LIN28B-long isoform. This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade , which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform..