Sarah F. Andres

Insulin receptor isoform switching in intestinal stem cells, progenitors, differentiated lineages and tumors: evidence that IR-B limits proliferation

Summary Despite 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 ApcMin/+ 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.

Loss of Stromal IMP1 Promotes a Tumorigenic Microenvironment in the Colon.

The colon tumor microenvironment 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 histologic grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in protumorigenic 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. Implications: The tumor-suppressive role of stromal IMP1 and its ability to modulate protumorigenic factors suggest that IMP1 status is important for the initiation and growth of epithelial tumors. Mol Cancer Res; 13(11); 1478–86. ©2015 AACR. See related article by Koltsova and Grivennikov, p. 1452

Deletion of intestinal epithelial insulin receptor attenuates high-fat diet-induced elevations in cholesterol and stem, enteroendocrine, and Paneth cell mRNAs

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.

Reduced Insulin-like Growth Factor I Receptor and Altered Insulin Receptor Isoform mRNAs in Normal Mucosa Predict Colorectal Adenoma Risk

Background: Hyperinsulinemia resulting from obesity and insulin resistance is associated with increased risk of many cancers, but the biology underlying this risk is unclear. We hypothesized that increased mRNA levels of the insulin-like growth factor I receptor (IGFIR) versus the insulin receptor (IR) or elevated ratio of IR-A:IR-B isoforms in normal rectal mucosa would predict adenoma risk, particularly in individuals with high body mass index (BMI) or plasma insulin. Methods: Biopsies from normal rectal mucosa were obtained from consenting patients undergoing routine colonoscopy at University of North Carolina Hospitals (Chapel Hill, NC). Subjects with colorectal adenomas were classified as cases (n = 100) and were matched to adenoma-free controls (n = 98) based on age, sex, and BMI. IGFIR and IR mRNA levels were assessed by qRT-PCR, and IR-A:IR-B mRNA ratios by standard PCR. Plasma insulin and crypt apoptosis were measured by ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. Logistic regression models examined relationships between receptor mRNAs, BMI, plasma insulin, and adenoma risk. Results: Unexpectedly, cases were significantly more likely to have lower IGFIR mRNA levels than controls. No overall differences in total IR mRNA or IR-A:IR-B ratios were observed between cases and controls. Interestingly, in patients with high plasma insulin, increased IR-A:IR-B ratio was associated with increased likelihood of having adenomas. Conclusions: Our work shows novel findings that reduced IGFIR mRNA and, during high plasma insulin, increased IR-A:IR-B ratios in normal rectal mucosa are associated with colorectal adenoma risk. Impact: Our work provides evidence supporting a link between IGFIR and IR isoform expression levels and colorectal adenoma risk. Cancer Epidemiol Biomarkers Prev; 23(10); 2093–100. ©2014 AACR.

Obesity and intestinal epithelial deletion of the insulin receptor, but not the IGF 1 receptor, affect radiation-induced apoptosis in colon

Current views suggest that apoptosis eliminates genetically damaged cells that may otherwise form tumors. Prior human studies link elevated insulin and reduced apoptosis to risk of colorectal adenomas. We hypothesized that hyperinsulinemia associated with obesity would lead to reduced colon epithelial cell (CEC) apoptosis after radiation and that this effect would be altered by deletion of the insulin-like growth factor (IGF) 1 receptor (IGF1R) or the insulin receptor (IR). Mice with villin-Cre-mediated IGF1R or IR deletion in CECs and floxed littermates were fed a high-fat diet to induce obesity and hyperinsulinemia or control low-fat chow. Mice were exposed to 5-Gy abdominal radiation to induce DNA damage and euthanized 4 h later for evaluation of apoptosis by localization of cleaved caspase-3. Obese mice exhibited decreased apoptosis of genetically damaged CECs. IGF1R deletion did not affect CEC apoptosis in lean or obese animals. In contrast, IR loss increased CEC apoptosis in both diet groups but did not prevent antiapoptotic effects of obesity. Levels of p53 protein were significantly reduced in CECs of obese mice with intact IR but increased in both lean and obese mice without IR. Levels of mRNAs encoding proapoptotic Perp and the cell cycle inhibitor Cdkn1b/p27 were reduced in CECs of obese mice and increased in lean mice lacking IR. Together, our studies provide novel evidence for antiapoptotic roles of obesity and IR, but not IGF1R, in colonic epithelium after DNA damage. However, neither IR nor IGF1R deletion prevented a reduction in radiation-induced CEC apoptosis during obesity and hyperinsulinemia.

Differential Regulation of LET-7 by LIN28B Isoform–Specific Functions

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 LET-7 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 LET-7–dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7. Coexpression of the short isoform in the LIN28B-long isoform–expressing cells rescued the phenotypes induced by the LIN28B-long isoform. Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, 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. Mol Cancer Res; 16(3); 403–16. ©2018 AACR.

Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem 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.

Posttranscriptional regulation of intestinal epithelial cell repair by RNA binding protein IMP1

RNA binding proteins, such as IMP1, are emerging as essential regulators of intestinal development and cancer. IMP1 hypomorphic mice exhibit severe intestinal growth defects, yet it’s role in adult intestinal epithelium is unclear. We employed ribosome profiling to test the effect of IMP1 loss on the “translatome” in colon cancer cell lines. In parallel, we evaluated mice with intestinal epithelial-specific Imp1 deletion (Imp1ΔIEC) following irradiation or colitis models. Ribosome-profiling revealed translation efficiency changes for multiple pathways important for intestinal homeostasis, including autophagy, in IMP1 knockout cells. We found increased autophagy flux in Imp1ΔIEC mice, reinforced through in silico and biochemical analyses revealing direct binding of IMP1 to autophagy transcripts MAP1LC3B and ATG3. We found that Imp1ΔIEC mice exhibit enhanced recovery following irradiation, which is attenuated with genetic deletion of autophagy gene Atg7. Finally, we demonstrated that IMP1 is upregulated in Crohn’s disease patients and Imp1 loss lessened colitis severity in mice. These studies demonstrate that IMP1 acts as a posttranscriptional regulator of gut epithelial repair post-irradiation and colitis, in part through modulation of autophagy.

The LIN28B–IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine

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 IMP1 as a principle node for gene expression regulation downstream from LIN28B 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.

The Molecular Basis of Metastatic Colorectal Cancer

Purpose of ReviewMetastatic colorectal cancer (CRC) is a vexing clinical problem. In contrast to early-stage disease, once CRC metastasizes to other organs, long-term survival is compromised. We seek to review the molecular pathogenesis, animal models, and functional genomics for an enhanced understanding of how CRC metastasizes and how this can be exploited therapeutically.Recent FindingsMouse models may recapitulate certain aspects of metastatic human CRC and allow for studies to identify regulators of metastasis. Modulation of transcription factors, onco-proteins, or tumor suppressors has been identified to activate known metastatic pathways. CD44 variants, microRNAs, and RNA binding proteins are emerging as metastatic modulators.SummaryCRC metastasis is a multi-faceted and heterogeneous disease. Despite common pathways contributing to metastatic development, there are numerous variables that modulate metastatic signals in subsets of patients. It is paramount that studies continue to investigate metastatic drivers, enhancers, and inhibitors in CRC to develop therapeutic targets and improve disease outcomes.