Basal expression of insulin‐like growth factor 1 receptor determines intrinsic resistance of cancer cells to a phosphatidylinositol 3‐kinase inhibitor ZSTK474

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3‐kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors (PI3Kis) remain unclear. We previously identified that overexpression of insulin‐like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long‐term exposure to PI3Kis. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug‐naïve cancer cells to PI3Kis and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R showed resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine‐phosphorylated insulin receptor substrate 1 at high levels, which was dependent on basal IGF1R expression. In these cells, the efficacy of ZSTK474 in vitro and in vivo was improved by its combination with the IGF1R inhibitor OSI‐906. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in an in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3Kis with IGF1R inhibitors for treating IGF1R‐positive human cancers.     

Insulin‐like growth factor 1 receptor expression in wild‐type GISTs: A potential novel therapeutic target

Aberrations of the Insulin‐like Growth Factor (IGF) system have been found in association with a variety of cancer types. The potential role of IGF1R has been postulated in a small subset of GISTs, but until now the implications of its aberrations have not been defined. The aim of the study was to examine the IGF1R status in patients with gastric GIST in regard to KIT and PDGFRA genotype. Fresh resection specimens were collected from 8 primary tumours [2 wild‐type (WT) and 6 mutant cases]. IGF1R was studied as gene expression profiling with Affymetrix GeneChip HG‐U133Plus 2.0 arrays and as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and at protein level with western blotting (WB) and immunohistochemistry (IHC). The unsupervised analysis of gene expression profiling of our patients merged with a data set from gastric GISTs identified 2 patients out of 8 with different expression of IGF1R. The data were confirmed by WB and IHC. In particular, IGF1R was upregulated in 2 young patients (<30‐years old), who had both WT disease and metastases at diagnosis. The SNP array analysis showed that none of the tumours had IGF1R amplification. GISTs are characterized by abnormalities of the KIT and PDGFRA receptors that affect prognosis and response to tyrosine kinase inhibitors. Both young adult with WT GIST had the over‐expression of IGF1R at mRNA and protein level. These results further confirm the hypothesis that IGF1R may be a potential therapeutic target in GISTs lacking KIT and PDGFRA mutations. © 2009 UICC     

Circulating insulin‐like growth factor peptides and prostate cancer risk: A systematic review and meta‐analysis

Insulin‐like growth factors (IGF‐I, IGF‐II) and their binding proteins (IGFBP‐1–6) play a key role in cell proliferation, differentiation and apoptosis, suggesting possible involvement in carcinogenesis. Several epidemiological studies show associations of IGFs with prostate cancer. We searched the published literature for all studies relating levels of IGFs or IGFBPs with prostate cancer. We performed random effects meta‐analysis to calculate summary odds ratios. The number of studies (prostate cancer cases) included in each meta‐analysis were 42 (7,481) IGF‐I; 10 (923) IGF‐II; 3 (485) IGFBP‐1; 5 (577) IGFBP‐2; 29 (6,541) IGFBP‐3 and 11 (3,545) IGF‐1:IGFBP‐3 ratio. The pooled odds ratios (95% confidence intervals) per standard deviation increase in peptide were: IGF‐I, OR = 1.21 (1.07, 1.36); IGF‐II, OR = 1.17 (0.93, 1.47); IGFBP‐1, OR = 1.21 (0.62, 2.33); IGFBP‐2, OR = 1.18 (0.90, 1.54); IGFBP‐3, OR = 0.88 (0.79, 0.98); IGFI:IGFBP‐3 ratio, OR = 1.10 (0.97, 1.24). For all exposures, there was substantial heterogeneity (all I² > 75%), partly explained by study design: the magnitude of associations was smaller in prospective vs. retrospective studies, and for IGFBP‐3, the inverse association with prostate cancer risk was seen in retrospective but not prospective studies. There was weak evidence that associations of IGF‐I and IGFBP‐3 with prostate cancer were stronger for advanced disease. Our meta‐analysis confirms that raised circulating lGF‐I is positively associated with prostate cancer risk. Associations between IGFBP‐3 and prostate cancer were inconsistent, and there was little evidence for a role of IGF‐II, IGFBP‐1 or IGFBP‐2 in prostate cancer risk. © 2008 Wiley‐Liss, Inc.     

Preclinical and first‐in‐human phase I studies of KW‐2450, an oral tyrosine kinase inhibitor with insulin‐like growth factor receptor‐1/insulin receptor selectivity

Numerous solid tumors overexpress or have excessively activated insulin‐like growth factor receptor‐1 (IGF‐1R). We summarize preclinical studies and the first‐in‐human study of KW‐2450, an oral tyrosine kinase inhibitor with IGF‐1R and insulin receptor (IR) inhibitory activity. Preclinical activity of KW‐2450 was evaluated in various in vitro and in vivo models. It was then evaluated in a phase I clinical trial in 13 patients with advanced solid tumors (NCT00921336). In vitro, KW‐2450 inhibited human IGF‐1R and IR kinases (IC₅₀ 7.39 and 5.64 nmol/L, respectively) and the growth of various human malignant cell lines. KW‐2450 40 mg/kg showed modest growth inhibitory activity and inhibited IGF‐1‐induced signal transduction in the murine HT‐29/GFP colon carcinoma xenograft model. The maximum tolerated dose of KW‐2450 was 37.5 mg once daily continuously; dose‐limiting toxicity occurred in two of six patients at 50 mg/day (both grade 3 hyperglycemia) and in one of seven patients at 37.5 mg/day (grade 3 rash). Four of 10 evaluable patients showed stable disease. Single‐agent KW‐2450 was associated with modest antitumor activity in heavily pretreated patients with solid tumors and is being further investigated in combination therapy with lapatinib/letrozole in patients with human epidermal growth factor receptor 2‐postive metastatic breast cancer.     

Increased expression of the mannose 6-phosphate/insulin-like growth factor-II receptor in breast cancer cells alters tumorigenic properties in vitro and in vivo

The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF-IIR) is thought to act as a suppressor of tumor growth by binding the mitogenic peptide IGF-II and modulating its extracellular levels via degradation. This receptor has been found to be absent or nonfunctional in a high proportion of breast tumors as a result of LOH and mutation of the gene. In our study, we have examined the effect of increasing expression of M6P/IGF-IIR on breast cancer cell tumorigenicity. MDA-MB-231 breast cancer cells stably transfected with M6P/IGF-IIR cDNA exhibited not only a greatly reduced ability to form tumors but also a markedly reduced growth rate in nude mice. In vitro, increased M6P/IGF-IIR expression resulted in 2-fold reduced uptake of IGF-II and was associated with reduced cellular invasiness and motility. Cells with increased M6P/IGF-IIR expression exhibited reduced phosphorylation of IGF-I receptor and p44/42 MAPK compared to vector transfectants, or wild-type MDA-MB-231 cells. These results therefore suggest that M6P/IGF-IIR levels can modulate breast cancer cell tumorigenicity by a mechanism that may involve altered IGF-I receptor signaling.     

Murine osteosarcoma primary tumour growth and metastatic progression is maintained after marked suppression of serum insulin‐like growth factor I

The insulin‐like growth factor I (IGF‐I) signaling pathway has been shown to play an important role in several aspects of cancer biology, including metastasis. The aim of this study was to define the contribution of serum (endocrine) and local (tumour microenvironment) IGF‐I on osteosarcoma tumour growth and metastasis, a cancer that is known to be dependent on the IGF‐I axis. To test this hypothesis, we evaluated the primary tumour growth and metastatic progression of K7M2 murine osteosarcoma cells injected to a genetically engineered mouse [liver‐specific IGF‐I deficient (LID)] in which serum IGF‐I levels are reduced by 75%, while maintaining expression of IGF‐I in normal tissues. We first demonstrated that IGF‐I in the tumour and the tumour‐microenvironment were maintained in the LID mice. Within this designed model, there was no difference in primary tumour growth or in pulmonary metastasis in LID mice compared to control mice. Furthermore, there was no difference in the number or localization of single metastatic cells immediately after their arrival in the lungs of LID mice and control mice, as analysed by single cell video microscopy. Collectively, these data suggest that marked reduction in serum IGF‐I is not sufficient to slow the progression of either primary or metastatic models of osteosarcoma. © 2008 Wiley‐Liss, Inc.     

Estrogen and insulin‐like growth factor 1 synergistically promote the development of lung adenocarcinoma in mice

Estrogen receptor (ER) and insulin‐like growth factor‐1 receptor (IGF‐1R) signaling are implicated in lung cancer progression. Based on their previous findings, the authors sought to investigate whether estrogen and IGF‐1 act synergistically to promote lung adenocarcinoma (LADE) development in mice. LADE was induced with urethane in ovariectomized Kunming mice. Tumor‐bearing mice were divided into seven groups: 17β‐estradiol (E2), E2+fulvestrant (Ful; estrogen inhibitor), IGF‐1, IGF‐1+AG1024 (IGF‐1 inhibitor), E2+IGF‐1, E2+IGF‐1+Ful+AG1024 and control groups. After 14 weeks, the mice were sacrificed, and then the tumor growth was determined. The expression of ERα/ERβ, IGF‐1, IGF‐1R and Ki67 was examined using tissue‐microarray‐immunohistochemistry, and IGF‐1, p‐ERβ, p‐IGF‐1R, p‐MAPK and p‐AKT levels were determined based on Western blot analysis. Fluorescence‐quantitative polymerase chain reaction was used to detect the mRNA expression of ERβ, ERβ2 and IGF‐1R. Tumors were found in 93.88% (46/49) of urethane‐treated mice, and pathologically proven LADE was noted in 75.51% (37/49). In the E2+IGF‐1 group, tumor growth was significantly higher than in the E2 group (p < 0.05), the IGF‐1 group (p < 0.05) and control group (p < 0.05). Similarly, the expression of ERβ, p‐ERβ, ERβ2, IGF‐1, IGF‐1R, p‐IGF‐1R, p‐MAPK, p‐AKT and Ki67 at the protein and/or mRNA levels was markedly higher in the ligand group than in the ligand + inhibitor groups (all p < 0.05). This study demonstrated for the first time that estrogen and IGF‐1 act to synergistically promote the development of LADE in mice, and this may be related to the activation of the MAPK and AKT signaling pathways in which ERβ1, ERβ2 and IGF‐1R play important roles.     

Insulin‐like growth factor‐1 receptor and phosphorylated AKT‐serine 473 expression in 132 resected thymomas and thymic carcinomas

BACKGROUND:

Thymic malignancies are rare tumors. The insulin‐like growth factor‐1 (IGF‐1)/IGF‐1 receptor (IGF‐1R) system is involved in the development of the thymus. IGF‐1R expression in thymic epithelial malignancies is unknown.

METHODS:

The authors investigated the expression of IGF‐1R and phosphorylated AKT serine 473 (p‐AKT) by using immunohistochemistry and examined the clinicopathologic correlations in a retrospective, single‐institution surgical series of 132 patients with thymic epithelial malignancies.

RESULTS:

Earlier disease stage, less aggressive histologic types, and complete resection were significant positive prognostic factors for disease‐related survival and progression‐free survival, and being a woman was a better prognostic factor for disease‐related survival. IGF‐1R and p‐AKT protein levels were expressed in 20% and 36% of thymic tumors, respectively. Both markers were expressed more commonly in recurrent disease than in primary tumors, in more aggressive subtypes, and in more advanced disease stages. There was a trend toward better survival and progression‐free survival in patients who were negative for IGF‐1R or p‐AKT expression in the whole series. When only the 91 primary tumors, IGF1R expression was associated with worse progression‐free survival (P < .001).

CONCLUSIONS:

The current retrospective analysis demonstrated that disease stage, tumor histology, sex, and resection type were major prognostic factors in the survival of patients with thymic malignancies. The expression levels of IGF‐1R and p‐AKT in thymic tumors suggested that IGF‐1R is a potential target for treatment. Cancer 2010. Published 2010 by the American Cancer Society.     

Serum insulin‐like,growth factor binding protein‐related protein 1 (IGFBP‐rP1) and endometrial cancer risk in Chinese women

Hyperinsulinemia and the metabolic syndrome confer increased risks of endometrial carcinoma. The roles of insulin, and, insulin‐like growth factor‐binding proteins (IGFBPs) in the etiology of endometrial carcinoma, remain unclear. We recruited 206 patients with endometrial carcinoma and 350 healthy women to a case–control study of fasting insulin and IGFBP‐related protein 1 (IGFBP‐rP1) in a Chinese tertiary centre. Patients with endometrial carcinoma had higher insulin concentrations (14.8 ± 16.7 vs. 8.1 ± 9.4 μU/mL; p < 0.001) and lower IGFBP‐rP1 levels (17.5 ± 17.2 vs. 22.4 ± 22.8 μg/L; p = 0.018) than controls. High insulin and IGFBP‐rP1 levels were both positively and negatively associated with endometrial cancer (odds ratio for the highest tertile versus the lowest tertile: insulin: 4.11; 95% CI = 2.61–6.47; IGFBP‐rP1: 0.38; 95% CI = 0.24–0.60). Logistic regression analysis confirmed the associations between endometrial carcinoma and fasting insulin or IGFBP‐rP1 after adjustments for age, BMI, serum glucose, cholesterol, triglycerides and high‐density lipoprotein cholesterol (odds ratio for the highest tertile versus the lowest tertile: insulin: 2.13; 95% CI = 1.30–3.49; IGFBP‐rP1: 0.57; 95% CI = 0.34–0.94). Hyperinsulinemia and high IGFBP‐rP1 levels confer altered risks for endometrial carcinoma.     

The mannose 6‐phosphate/insulin‐like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells

The mannose 6‐phosphate/insulin‐like growth factor II receptor (M6P/IGF2R) mediates biosynthetic sorting and endocytosis of various factors that impinge on the proliferation, migration and invasiveness of tumour cells. The gene encoding M6P/IGF2R is frequently lost or mutated in a wide range of malignant tumours including squamous cell carcinomas. We have previously shown that M6P/IGF2R‐deficient SCC‐VII murine squamous cell carcinoma cells secrete large amounts of pro‐invasive lysosomal proteinases. Furthermore, the formation of mature lysosomes is impaired in SCC‐VII cells. To assess the link between M6P/IGF2R status and tumour invasion, we have now generated SCC‐VII lines stably transfected with human M6P/IGF2R cDNA. Reconstitution of functional M6P/IGF2R expression in SCC‐VII cells strongly improves the intracellular retention of lysosomal proteinases and restores the formation of mature lysosomes. In addition, the presence of heterologous M6P/IGF2R compromises the growth of SCC‐VII cells both in vitro and in vivo. Remarkably, M6P/IGF2R expression also reduces the invasive capacity of SCC‐VII cells in response to various chemoattractants. These results indicate that the M6P/IGF2R status influences the metastatic propensity of squamous cell carcinomas. © 2008 Wiley‐Liss, Inc.     

Insulin‐like growth factor‐1 receptor as a novel prognostic marker and its implication as a cotarget in the treatment of human adenocarcinoma of the esophagus

Insulin‐like growth factor‐1 receptor (IGF‐1R) and human epidermal growth factor receptor‐2 (HER2) receptor expression has been found to be a key regulator of tumorigenesis. The purpose of our study was to establish the prognostic significance of IGF‐1R in esophageal cancer and to determine the effect of IGF‐1R and HER2 targeting with α‐IR3 and Herceptin? antibodies on the proliferation of esophageal cancer cells in vitro. IGF‐1R expression and clinicopathological correlations were analyzed with a tissue microarray containing 234 esophageal cancer specimens (133 adenocarcinomas and 101 squamous cell carcinomas). Proliferation changes associated with Herceptin? and α‐IR3 blockage were evaluated with the unique human esophageal cancer cell lines Pt1590 and LN1590. IGF‐1R and HER2 expression levels, activation and phosphorylation status of downstream signaling proteins involved in the activation pathways were analyzed by Western blotting. IGF‐1R overexpression was detected in 121 (52%) of the 234 esophageal tumors examined. In the subgroup of 87 HER2‐positive tumors, 93.1% showed concordant overexpression for IGF‐1R. IGF‐1R was identified as a variable associated with reduced overall survival for adenocarcinoma (p = 0.05), but not for squamous cell carcinoma. The combination of Herceptin? and α‐IR3 was more effective in inhibiting in vitro proliferation than treatment with either agent alone (p < 0.01). This was associated with a decrease in HER2 and IGF‐1R protein levels and suppression of Akt‐ and MAP kinase phosphorylation. IGF‐1R expression can be used as a novel prognostic marker for adenocarcinomas of the esophagus. Cotreatment with IGF‐1R and HER2 antibodies might become a valuable and effective treatment option in esophageal adenocarcinoma.     

Energy restriction at young age,genetic variants in the insulin‐like growth factor pathway and colorectal cancer risk in the Netherlands Cohort Study

The energy restriction (ER)‐colorectal cancer (CRC) association is inconsistent in literature. To strengthen the biological plausibility of the ER‐CRC association, we investigated whether genetic variation in the insulin‐like growth factor (IGF) pathway, a putative underlying mechanism, modulated this association in the Netherlands Cohort Study. Participants completed a questionnaire (n = 120,852) and provided toenail clippings for DNA (～75%) at baseline. Individuals living in a Western city during the Hunger Winter (1944–45) or Western rural versus non‐Western area were exposed to (severe) ER at young age. Genotyping was performed for 3,768 subcohort members and 2,580 CRC cases (case‐cohort with 16.3 years follow‐up). Cox hazard ratios for CRC were estimated across combined categories of ER and a genetic sum score of unfavorable alleles based on 18 single nucleotide polymorphisms in IGF‐related genes and ER and an IGF1 19‐CA repeat polymorphism. The reference included ER exposed individuals, so that increased hazard ratios were expected in higher combined categories for calculating relative excess risks due to interaction (additive interactions). Wald tests for multiplicative interactions were also performed. Multiplicative and additive interactions were nonsignificant. Combined ER‐genetic sum score categories showed increasing CRC risks in men, but confidence intervals were wide. Women carrying two variant IGF1 19‐CA repeat alleles versus those carrying two wild type IGF1 19‐CA repeat alleles were at an ～50% decreased CRC risk, irrespective of ER exposure. In conclusion, data indicate that the IGF pathway might be involved in the ER‐CRC association in men, but not women, although interactions were nonsignificant, hampering definite conclusions.     

Assessing the role of insulin‐like growth factors and binding proteins in prostate cancer using Mendelian randomization: Genetic variants as instruments for circulating levels

Circulating insulin‐like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome‐wide association study [GWAS] and 48 in reported candidate genes). In ～700 men without prostate cancer and two replication cohorts (N ～ 900 and ～9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF‐I, IGF‐II, IGFBP‐2 and IGFBP‐3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (< 7) vs. high (≥ 7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF‐II and IGFBP‐3, less so for IGF‐I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF‐II/IGFBP‐3 level‐raising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF‐II (～265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker.     

胰岛素样生长因子l及其受体对子宫肌瘤生长的影响

目的 测定胰岛素样生长因子1(IGF 1)及其受体(IGF 1R)在子宫肌瘤患者的子宫平滑肌瘤组织、血清中的表达,旨在探讨IGF 1、IGF 1R在子宫肌瘤发病机制中的作用。方法 采用酶联免疫吸附实验(ELISA)方法,测定26例子宫肌瘤患者子宫平滑肌瘤、正常子宫平滑肌组织及血清中IGF1、IGF 1R的表达,每例患者充当组织测定的自身对照,选择30例健康妇女作为血清测定的对照组。结果 ①子宫平滑肌瘤组织中IGF 1、IGF 1R的表达较正常子宫平滑肌增强,进行统计学分析,两组差异有显著性(P<0.01)。②子宫肌瘤患者血清中IGF-1表达与对照组差异无显著性(P>0.05);IGF1R表达较对照组增强,两组差异有显著性(P<0.01)。结论 由此推测子宫平滑肌瘤组织中IGF 1、IGF 1R过度表达可能是子宫肌瘤发生的机制之一,IGF 1主要以自分泌、旁分泌作用方式促进肌瘤的生长,IGF 1可能是子宫肌瘤生长的调节因子。     

A recombinant humanized anti-insulin-like growth factor receptor type I antibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts

Interaction of insulin-like growth factor receptor I (IGF-IR) with its ligands has been reported to induce cell proliferation, transformation and blockade of cell apoptotic functions. IGF-IR is overexpressed on numerous tumor cell types and its blockade could be of importance for anti-cancer therapy. We have generated a humanized anti-IGF-IR antibody h7C10 that blocks in vitro IGF-I and IGF-II-induced cell proliferation of MCF-7 breast cancer cells. Analysis of the IGF-I transduction cascade demonstrated that the humanized anti-IGF-IR antibody and its murine parental form block IGF-I-induced tyrosine phosphorylation, both its beta-chain and IRS-1 tyrosine phosphorylation. This presumably leads to cell cycle arrest and, consequently, growth inhibition. Treatment of nude mice bearing either human breast cancer cells (MCF-7) or non small lung cancer cells (A549) with h7C10, or its murine parental form 7C10, inhibited significantly tumor growth. An almost complete inhibition of A549 tumor growth was observed when mice were treated with the anti-IGF-IR antibody combined with either a chemotherapeutic agent, Vinorelbine or an anti-epidermal growth factor receptor (EGFR) antibody, 225. Combined therapy prolonged significantly the life span of mice in an orthotopic in vivo model of A549; the combination of the anti-IGF-IR antibody with an anti-EGFR antibody was superior to the Vinorelbine combination. The present results indicate that the humanized anti-IGF-IR antibody h7C10 has a great potential for cancer therapy when combined with either a chemotherapeutic agent or an antibody that targets other growth factor receptors, such as the epidermal growth factor receptor.     

A bispecific transmembrane antibody simultaneously targeting intra‐ and extracellular epitopes of the epidermal growth factor receptor inhibits receptor activation and tumor cell growth

Epidermal growth factor receptor (EGFR) plays an important role in essential cellular processes such as proliferation, survival and migration. Aberrant activation of EGFR is frequently found in human cancers of various origins and has been implicated in cancer pathogenesis. The therapeutic antibody cetuximab (Erbitux) inhibits tumor growth by binding to the extracellular domain of EGFR, thereby preventing ligand binding and receptor activation. This activity is shared by the single chain antibody fragment scFv(225) that contains the same antigen binding domain. The unrelated EGFR‐specific antibody fragment scFv(30) binds to the intracellular domain of the receptor and retains antigen binding upon expression as an intrabody in the reducing environment of the cytosol. Here, we used scFv(225) and scFv(30) domains to generate a novel type of bispecific transmembrane antibody termed 225.TM.30, that simultaneously targets intra‐ and extracellular EGFR epitopes. Bispecific 225.TM.30 and related membrane‐anchored monospecific 225.TM and TM.30 proteins carrying extracellular scFv(225) or intracellular scFv(30) antibody fragments linked to a transmembrane domain were expressed in EGFR‐overexpressing tumor cells using a doxycycline‐inducible retroviral system. Induced expression of 225.TM.30 and 225.TM, but not TM.30 reduced EGFR surface levels and ligand‐induced EGFR activation, while all three molecules markedly inhibited tumor cell growth. Co‐localization of 225.TM with EGFR was predominantly found on the cell surface, while interaction with 225.TM.30 and TM.30 proteins resulted in the redistribution of EGFR to perinuclear compartments. Our data demonstrate functionality of this novel type of membrane‐anchored intrabodies in tumor cells and suggest distinct modes of action of mono‐ and bispecific variants.     

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), a putative breast tumor suppressor gene

Loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor gene locus (M6P/IGF2R) on 6q26-27 has recently been demonstrated in approximately 30% of both invasive and in situ breast cancers. LOH was coupled with somatic point mutations in the remaining allele in several instances, leading to the proposition that M6P/IGF2R is a tumor suppressor gene [1]. Somatic mutations in M6P/IGF2R have also been described in hepatoma [2] and gastrointestinal cancers with the replication error positive (RER+) phenotype [3]. These data indicate that M6P/IGF2R loss of function mutations may be involved in the pathogenesis of a wide spectrum of malignancies. Extensive data on the normal function of the M6P/IGF2R suggest that loss of M6P/IGF2R activity may contribute to multiple aspects of tumor pathophysiology, including deregulated growth, apoptosis, angiogenesis and invasion.