Heterodimerization of Glycosylated Insulin-Like Growth Factor-1 Receptors and Insulin Receptors in Cancer Cells Sensitive to Anti-IGF1R Antibody

Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells

The N-recognin UBR4 of the N-end rule pathway is required for neurogenesis and homeostasis of cell surface proteins.

The N-end rule pathway is a proteolytic system in which single N-terminal amino acids of proteins act as a class of degrons (N-degrons) that determine the half-lives of proteins. We have previously identified a family of mammals N-recognins (termed UBR1, UBR2, UBR4/p600, and UBR5/EDD) whose conserved UBR boxes bind N-degrons to facilitate substrate ubiquitination and proteasomal degradation via the ubiquitin-proteasome system (UPS). Amongst these N-recognins, UBR1 and UBR2 mediate ubiquitination and proteolysis of short-lived regulators and misfolded proteins. Here, we characterized the null phenotypes of UBR4-deficient mice in which the UBR box of UBR4 was deleted. We show that the mutant mice die around embryonic days 9.5-10.5 (E9.5-E10.5) associated with abnormalities in various developmental processes such as neurogenesis and cardiovascular development. These developmental defects are significantly attributed to the inability to maintain cell integrity and adhesion, which significantly correlates to the severity of null phenotypes. UBR4-loss induces the depletion of many, but not all, proteins from the plasma membrane, suggesting that UBR4 is involved in proteome-wide turnover of cell surface proteins. Indeed, UBR4 is associated with and required to generate the multivesicular body (MVB) which transiently store endocytosed cell surface proteins before their targeting to autophagosomes and subsequently lysosomes. Our results suggest that the N-recognin UBR4 plays a role in the homeostasis of cell surface proteins and, thus, cell adhesion and integrity

Urinary Proteome Profile Predictive of Disease Activity in Rheumatoid Arthritis

Current serum biomarkers for rheumatoid arthritis (RA) are not highly sensitive or specific to changes of disease activities. Thus, other complementary biomarkers have been needed to improve assessment of RA activities. In many diseases, urine has been studied as a window to provide complementary information to serum measures. Here, we conducted quantitative urinary proteome profiling using liquid chromatographytandem mass spectrometry (LCMS/MS) and identified 134 differentially expressed proteins (DEPs) between RA and osteoarthritis (OA) urine samples. By integrating the DEPs with gene expression profiles in joints and mononuclear cells, we initially selected 12 biomarker candidates related to joint pathology and then tested their altered expression in independent RA and OA samples using enzyme-linked immunosorbent assay. Of the initial candidates, we selected four DEPs as final candidates that were abundant in RA patients and consistent with those observed in LCMS/MS analysis. Among them, we further focused on urinary soluble CD14 (sCD14) and examined its diagnostic value and association with disease activity. Urinary sCD14 had a diagnostic value comparable to conventional serum measures and an even higher predictive power for disease activity when combined with serum C-reactive protein. Thus, our urinary proteome provides a diagnostic window complementary to current serum parameters for the disease activity of RA.1

Soluble Fas ligand drives autoantibody-induced arthritis by binding to DR5/TRAIL-R2

To date, no study has demonstrated that soluble Fas ligand (sFasL)-mediated inflammation is regulated via interaction with Fas in vivo. We found that FasL interacts specifically with tumor necrosis factor receptor superfamily (TNFRSF)10B, also known as death receptor (DR)5. Autoantibody-induced arthritis (AIA) was attenuated in FasL (Fasl(gld/gld))- and soluble FasL (Fasl(Delta s/Delta s))-deficient mice, but not in Fas (Fas(lpr/lpr) and Fas(-/-))- or membrane FasL (Fasl(Delta m/Delta m))-deficient mice, suggesting sFasL promotes inflammation by binding to a Fas-independent receptor. Affinity purification mass spectrometry analysis using human (h) fibroblast-like synovial cells (FLSCs) identified DR5 as one of several proteins that could be the elusive Fas-independent FasL receptor. Subsequent cellular and biochemical analyses revealed that DR5 interacted specifically with recombinant FasL-Fc protein, although the strength of this interaction was approximately 60-fold lower than the affinity between TRAIL and DR5. A microarray assay using joint tissues from mice with arthritis implied that the chemokine CX3CL1 may play an important downstream role of the interaction. The interaction enhanced Cx3cl1 transcription and increased sCX3CL1 production in FLSCs, possibly in an NF-kappa B-dependent manner. Moreover, the sFasL-DR5 interaction-mediated CX3CL1-CX3CR1 axis initiated and amplified inflammation by enhancing inflammatory cell influx and aggravating inflammation via secondary chemokine production. Blockade of FasL or CX3CR1 attenuated AIA. Therefore, the sFasL-DR5 interaction promotes inflammation and is a potential therapeutic target.Y

Urinary Proteome Profile Predictive of Disease Activity in Rheumatoid Arthritis

Current serum biomarkers for rheumatoid arthritis (RA) are not highly sensitive or specific to changes of disease activities. Thus, other complementary biomarkers have been needed to improve assessment of RA activities. In many diseases, urine has been studied as a window to provide complementary information to serum measures. Here, we conducted quantitative urinary proteome profiling using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and identified 134 differentially expressed proteins (DEPs) between RA and osteoarthritis (OA) urine samples. By integrating the DEPs with gene expression profiles in joints and mononuclear cells, we initially selected 12 biomarker candidates related to joint pathology and then tested their altered expression in independent RA and OA samples using enzyme-linked immunosorbent assay. Of the initial candidates, we selected four DEPs as final candidates that were abundant in RA patients and consistent with those observed in LC–MS/MS analysis. Among them, we further focused on urinary soluble CD14 (sCD14) and examined its diagnostic value and association with disease activity. Urinary sCD14 had a diagnostic value comparable to conventional serum measures and an even higher predictive power for disease activity when combined with serum C-reactive protein. Thus, our urinary proteome provides a diagnostic window complementary to current serum parameters for the disease activity of RA

Identification of a specific N-linked glycosylation site (N913) of IGF1R in sensitive cell lines and its functional importance in the response to figitumumab.

<p>A) Identification of the NLG site occupancy of IGF1Rβ subunits. IGF1Rβ subunits containing N-linked glycosylation sites (Asn747, Asn756, Asn764, Asn900, and Asn913) were isolated from both drug sensitive (SNU719, HepG2 and SNU368) and resistance (SNU638 and SNU354) cells and identified by tandem MS by an increase of 1.0 Da from the corresponding mass of Asn as a result of conversion from N-linked glycosylated Asn to Asp. All the NLG at Asn900 in both sensitive and resistance cells were determined to be occupied with N-glycosylation (filled rectangle). NLG at Asn913 of the sensitive cell lines (HepG2, SNU719, and SNU368) were determined to be occupied with N-glycosylation (filled rectangle), whereas N-glycosites at Asn913 of the resistance cell lines (SNU638 and SNU354) were found to be unoccupied with N-glycosylation. (open rectangle). B) Effect of the N913Q site mutation on electrophoretic mobility patterns of IGF1Rβ. Huh7 cells (an IGF1R-negative cell line) were transfected with the empty pcDNA3.1(-) expression vector(Control), pcDNA3.1(-) containing wild-type IGF1R cDNA (IGF1R WT), or pcDNA3.1(-) with IGF1R mutation type cDNA (IGF1R N913Q). An equal amount of the cell lysate from the transfected cells was then subjected to Western blot analysis for IGF1Rβ. C) Effect of N913Q site mutation on the formation of IGF1R/IR heterodimeric receptors. An equal amount of the cell lysate from transfected cells was then subjected to immunoprecipitation (IP) with anti-IGF1R antibody followed by Western blot analysis for IRβ and IGF1Rβ. Input = total cell lysate without IP. D) Effect of N913Q site mutation on IGF1R localization. An immunofluoresence assay was conducted to observe the localization of IGF1R. IGF1R reactivity was visualized by confocal laser scanning microscopy (Scale bar: 30 µm). Representative images are shown. Green: IGF1R, Blue: nuclei. E) Effect of N913Q site mutation on figitumumab sensitivity. Huh7 cells transfected with empty pcDNA3.1(-) vector, vector containing wild-type IGF1R cDNA, or vector containing IGF1R (N913Q) mutation type cDNA were plated in 96-well plates and treated with increasing concentrations of figitumumab for 120 h (left). Cell viability percentages with 1 µg/mL figitumumab (right). Six replicate wells were included in each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of remaining cells. * <i>P-</i>values <0.05; ** <i>P-</i>values <0.01.</p

Anti-proliferative effect of figitumumab.

<p>A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G₀/G₁, S, and G₂/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *<i>P-</i>values <0.05, **<i>P-</i>values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×10⁷) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm³. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student’s <i>t</i> test. *<i>P-</i>values <0.05; **<i>P-</i>values <0.01 versus control.</p

Anti-proliferative effect of figitumumab in gastric cancer and hepatocellular carcinoma cells.

<p>NOTE: The IC₃₀ values of figitumumab were determined by MTT assays, </p><p>The IC₃₀ value is the drug concentration required for 30% cell proliferation inhibition.</p><p>GC = gastric cancer; HCC = hepatocellular carcinoma.</p