Gemma V. Brierley

Differential Activation of Insulin Receptor Substrates 1 and 2 by Insulin-Like Growth Factor-Activated Insulin Receptors

ABSTRACT The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.

Butyrate-Induced Apoptosis in HCT116 Colorectal Cancer Cells Includes Induction of a Cell Stress Response

Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines.

A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR–directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1–104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (KD value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor–binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 Å. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I–induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR.

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test. Principal Findings In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2). Conclusions Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.

Silencing of the Insulin Receptor Isoform A Favors Formation of Type 1 Insulin-Like Growth Factor Receptor (IGF-IR) Homodimers and Enhances Ligand-Induced IGF-IR Activation and Viability of Human Colon Carcinoma Cells

Insulin receptor (IR) overexpression is common in cancers, with expression of the A isoform (IR-A, exon 11-) predominating over the B isoform. The IR-A signals a proliferative, antiapoptotic response to IGF-II, which itself can be secreted by tumors to establish an autocrine proliferative loop. Therefore, IGF-II signaling via the IR-A could mediate resistance to type 1 IGF receptor (IGF-IR) inhibitory drugs that are currently in development. This study addressed the role of the IR-A, using a small interfering RNA-based approach in SW480 human colon adenocarcinoma cells that coexpress the IGF-IR. Clonogenic survival was inhibited by depletion of the IGF-IR but not the IR-A, and dual receptor depletion had no greater effect than IGF-IR knockdown alone, suggesting that the IR-A could not compensate for IGF-IR loss. IGF-IR knockdown also resulted in a decrease in viability, whereas IR-A depletion resulted in increased viability. Consistent with this, upon IR-A depletion, we found a concomitant enhancement of IGF-IR activation by IGF-I and IGF-II, reduced formation of IGF-IR:IR-A hybrid receptors and increased IGF-IR homodimer formation. Together, these results suggest that IGF bioactivity is mediated more effectively by the IGF-IR than by the IR-A or receptor hybrids and that signaling via the IGF-IR is dominant to the IR-A in colon cancer cells that express both receptors.

Claudin-1 Expression Is Elevated in Colorectal Cancer Precursor Lesions Harboring the BRAF V600E Mutation.

BACKGROUND: Sessile serrated adenomas/polyps (SSA/P) are now recognised precursors of colorectal cancer (CRC) including cancers harbouring somatic BRAF (V600E) mutations. While the morphological diagnostic criteria of SSA/P have been established, distinguishing between small/early SSA/P and microvesicular hyperplastic polyps (MVHP) is challenging and may not be possible in routine practice. METHODS: Gene expression profiling of MVHP (n=5, all BRAF V600E wild-type) and SSA/P (n=5, all BRAF V600E mutant) samples was performed. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and immunohistochemical analysis was performed to verify the expression of claudin 1 (CLDN1) in MVHP and SSA/P. RESULTS: Gene expression profiling studies conducted between MVHP and SSA/P identified CLDN1 as the most statistically significant differentially expressed gene (p<0.05). Validation with qRT-PCR confirmed an up-regulation of CLDN1 in BRAF V600E mutant polyps regardless of polyp type (p<0.0005). Immunohistochemical analysis of CLDN1 expression in BRAF V600E mutant SSA/Ps (n=53) and MVHPs (n=111) and BRAF wild-type MVHPs (n=58), demonstrated a strong correlation between CLDN1 expression and the BRAF V600E mutation in both SSA/P and MVHP samples when compared to wild-type polyps (p<0.0001). CONCLUSION: This study demonstrates an up regulation of CLDN1 protein in serrated colorectal polyps including MVHP harbouring the BRAF V600E mutation. Our results demonstrated an apparent heterogeneity on the molecular level within the MVHP group and suggest that MVHP with somatic BRAF V600E mutation and up-regulated expression of CLDN1 are closely related to SSA/P and may in fact represent a continuous spectrum of the same neoplastic process within the serrated pathway of colorectal carcinogenesis.

Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR

OBJECTIVE Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms. DESIGN Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. RESULTS The IGF-IICI(N) analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICI(N) to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. CONCLUSIONS Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

A combined free-flow electrophoresis and DIGE approach to identify proteins regulated by butyrate in HT29 cells

Many biologically active agents exert a pleiotropic response in cells and tissues. This presents challenges in descriptive and comparative analysis of the proteome in response to these agents. Although free‐flow electrophoresis has been applied in a number of proteomic studies as a protein separation technique, the combination of free‐flow electrophoresis and DIGE has not yet been investigated for comparative proteomic analysis. In this study, we have compared the effects of butyrate on HT29 colorectal cancer cells with a particular focus on apoptosis and describe the utility of a novel approach combining free‐flow electrophoresis with DIGE to identify differentially expressed proteins. We verify the results obtained by the combined free‐flow electrophoresis and DIGE approach with Western blot analysis of selected proteins. We also report for the first time the regulation of a number of proteins by butyrate in HT29 colorectal cells including peptidyl‐prolyl cis‐trans isomerase A (cyclophilin A) and profilin‐1.

Analysis of 32 Blood-Based Protein Biomarkers for their Potential to Diagnose Colorectal Cancer

Colorectal cancer (CRC) is largely viewed as a preventable disease but the prevalence is increasing worldwide. Although many faecal and blood-based biomarkers have been proposed as potential diagnostic markers, none have been successful in large cohort studies. In this study, ELISA was used to evaluate 32 candidate protein biomarkers in a single cohort of CRC patients (n=95) and age/sex matched controls (n=50). Of these, 12 markers differed statistically between cases and controls. Receiver operating characteristic analysis identified IL8, Mac2BP, TIMP1, and OPN as the best performing markers for overall CRC diagnosis. However, further analysis determined that IL6, TGFB1, TIMP2 and IGF2 were most accurate at identifying early stage disease. We also assessed the correlation between markers and determined that the strongest correlations existed between VEGFA and TGFB1 (r=0.65, p<0.0001), M30 and M65 (r=0.59, p<0.001), and between TGFB1 and TIMP1 (r=0.55, p<0.0001). This analysis provides a consistent baseline for identifying a potential panel of diagnostic protein biomarkers in blood. Our results highlight protein biomarker combinations that reflect the disease process and which may provide the sensitivity and specificity required a reliable diagnosis of CRC.

Circulating Levels of the Wnt Antagonist Dkk-3 as a Diagnostic Marker for Colorectal Cancer

The Wnt antagonist Dickkopf-3 (Dkk-3) has been implicated in several stages of tumour development in a wide range of human cancers, including colorectal cancer (CRC). However, the usefulness of serum Dkk-3 levels as a diagnostic biomarker for CRC has yet to be determined. In this study we used an ELISA immunoassay to examine serum Dkk-3 protein levels in a retrospective cohort of CRC patients (n = 89) and age, gender matched controls (n = 46). The median concentration of Dkk-3 was significantly (p = 0.0003) lower in CRC patient serum samples (29.3 ng/ml, range 10.4 – 67.8 ng/ml) when compared to control serum samples (36.8 ng/ml, range 20.7 – 67.4 ng/ml). Receiver operating characteristic analysis demonstrated at 90% specificity, serum Dkk-3 levels distinguished CRC patients with 36% sensitivity (AUC = 0.69, 95% CI 0.60 – 0.78).