Randy L. Jirtle

Elevated plasma transforming growth factor-β1 levels in breast cancer patients decrease after surgical removal of the tumor

ObjectiveThe authors determined whether untreated breast cancer patients have elevated plasma levels of transforming growth factor-β1, (TGF-β1). Summary Background DataIncreased plasma TGF-β1 levels recently were found after chemotherapy in patients with advanced breast cancer. However, it currently is unknown whether this elevation in plasma TGF-β1 is caused by chemotherapy-induced normal tissue damage or whether it results from the presence of the tumor. MethodsAn enzyme-linked immunosorbent assay was used to measure plasma TGF-β1 levels in 26 newly diagnosed breast cancer patients before and after definitive surgery. Patients were grouped by postoperative tumor status: 1) negative lymph nodes (group 1); 2) positive lymph nodes (group 2); and 3) overt residual disease (group 3). The site of TGF-β1 production in the tumors was localized by immunohistochemistry and in situ hybridization. ResultsPlasma TGF-β1 levels were elevated preoperatively in 81% of the patients; TGF-β2 and TGF-β3 were undetectable. The preoperative TGF-β1, levels in the three patient groups were similar; however, the postoperative plasma TGF-β1 levels differed by disease status. The mean plasma TGF-β1 level in group 1 (n = 12) normalized after surgery (19.3 ± 3.2 vs. 5.5 ± 1.0 ng/mL, p < 0.001). In contrast, the mean plasma TGF-β1 levels remained above normal in both group 2 (n = 9) and group 3 (n = 5) after surgery. Transforming growth factor-β1 expression was found to be preferentially increased in the tumor stroma. ConclusionsBreast tumors result in increased plasma TGF-β1 levels in 81% of patients. After surgical removal of the primary tumor, the plasma TGF-β1 level normalizes in the majority of patients; persistently elevated levels correlate with the presence of lymph node metastases or overt residual tumor.

Disruption of Ligand Binding to the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor by Cancer-associated Missense Mutations

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) carries out multiple regulatory and transport functions, and disruption of IGF2R function has been implicated as a mechanism to increase cell proliferation. Several missense IGF2R mutations have been identified in human cancers, including the following amino acid substitutions occurring in the extracytoplasmic domain of the receptor: Cys-1262 → Ser, Gln-1445 → His, Gly-1449 → Val, Gly-1464 → Glu, and Ile-1572 → Thr. To determine what effects these mutations have on IGF2R function, mutant and wild-type FLAG epitope-tagged IGF2R constructs lacking the transmembrane and cytoplasmic domains were characterized for binding of insulin-like growth factor (IGF)-II and a mannose 6-phosphate-bearing pseudoglycoprotein termed PMP-BSA (where PMP is pentamannose phosphate and BSA is bovine serum albumin). The Ile-1572 → Thr mutation eliminated IGF-II binding while not affecting PMP-BSA binding. Gly-1449 → Val and Cys-1262 → Ser each showed 30–60% decreases in the number of sites available to bind both 125I-IGF-II and125I-PMP-BSA. In addition, the Gln-1445 → His mutant underwent a time-dependent loss of IGF-II binding, but not PMP-BSA binding, that was not observed for wild type. In all, four of the five cancer-associated mutants analyzed demonstrated altered ligand binding, providing further evidence that loss of IGF2R function is characteristic of certain cancers.

Transforming growth factor-beta receptors and mannose 6-phosphate/insulin-like growth factor-II receptor expression in human hepatocellular carcinoma.

ObjectiveThe authors examined the expression of transforming growth factor-beta receptor (TGF-βr) types I and II and the mannose 6-phosphate/insulin-like growth factor-II receptor (M6-P/IGF-IIr) in human hepatocellular carcinoma (HCC). Summary Background DataTransforming growth factor-beta (TGF-β) is part of a superfamily of peptide-signaling molecules that play an important role in modulating cell growth. It is secreted as a latent complex and therefore, must be activated to elicit a biological response. Bioactivaton of the TGF-β complex is facilitated by binding to the M6-P/IGF-IIr. Once activated, TGF-β exerts its effects by binding to specific cell membrane TGF-β receptors. The loss of responsiveness of hepatocytes to TGF-β has been implicated in hepatocarcinogenesis and could result from a loss in the expression of either the TGF-β receptors or the M6-P/IGF-IIr. MethodsHuman hepatocellular carcinomas and surrounding normal tissue were collected from operating room samples and snap-frozen in liquid nitrogen (n = 13). Tissues from two tumors were fixed in Omni-fix for sectioning and immunohistochemistry staining for the M6-P/IGF-IIr and TGF-β1. RNA was extracted from both normal and malignant liver tissue and analyzed using an RNase protection assay. SDS-PAGE of purified membrane hybridized with 125I-IGF-β1 and 125I-IGF-II was used to determine the TGF-β type I (TGF-βrl) and type II (TGF-βrll) receptors and M6-P/IGF-IIr protein levels, respectively. Gels were quantitated by phosphorimager, and a paired t test was used for statistical analysis. ResultsIn HCC, a 60% (p < 0.01) and 49% (p < 0.02) reduction in the mRNA levels for Tβrl and Tβrll, respectively, relative to the receptor levels in surrounding normal liver, was shown. A similar decrease in the receptor protein levels also was observed. The M6-P/IGF-IIr mRNA and protein

M6P/IGF2R loss of heterozygosity in head and neck cancer associated with poor patient prognosis

BackgroundThe mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes for a multifunctional receptor involved in lysosomal enzyme trafficking, fetal organogenesis, cytotoxic T cell-induced apoptosis and tumor suppression. The purpose of this investigation was to determine if the M6P/IGF2R tumor suppressor gene is mutated in human head and neck cancer, and if allelic loss is associated with poor patient prognosis.MethodsM6P/IGF2R loss of heterozygosity in locally advanced squamous cell carcinoma of the head and neck was assessed with six different gene-specific nucleotide polymorphisms. The patients studied were enrolled in a phase 3 trial of twice daily radiotherapy with or without concurrent chemotherapy; median follow-up for surviving patients is 76 months.ResultsM6P/IGF2R was polymorphic in 64% (56/87) of patients, and 54% (30/56) of the tumors in these informative patients had loss of heterozygosity. M6P/IGF2R loss of heterozygosity was associated with a significantly reduced 5 year relapse-free survival (23% vs. 69%, p = 0.02), locoregional control (34% vs. 75%, p = 0.03) and cause specific survival (29% vs. 75%, p = 0.02) in the patients treated with radiotherapy alone. Concomitant chemotherapy resulted in a better outcome when compared to radiotherapy alone only in those patients whose tumors had M6P/IGF2R loss of heterozygosity.ConclusionsThis study provides the first evidence that M6P/IGF2R loss of heterozygosity predicts for poor therapeutic outcome in patients treated with radiotherapy alone. Our findings also indicate that head and neck cancer patients with M6P/IGF2R allelic loss benefit most from concurrent chemotherapy.

The tetratricopeptide repeat containing Tg737 gene is a liver neoplasia tumor suppressor gene

The Tg737 gene was investigated for gross alterations in a series of rodent/human liver tumors and human tumorigenic cell lines. The Tg737 gene was found to be altered in approximately 40% of the rodent chemically-induced liver tumors, 40% of the human liver tumors, and in liver, kidney and pancreatic human tumor cell lines. Ectopic re-expression of the Tg737 gene in a Tg737 deleted mouse liver tumor cell line resulted in suppression of tumorigenic growth, without altering in vitro cell culture growth. Treatment of mice which are either homozygous normal or heterozygous deleted at the Tg737 locus with the carcinogen diethylnitrosamine resulted in an increase in preneoplastic foci formation in the Tg737 heterozygous deleted mice. Ectopic expression of the Tg737 gene results in multinucleated cells, loss of Tg737 gene expression results in the proliferation of liver stem cells (oval cells) without concomitant differentiation, and reexpression of the Tg737 gene reestablished responsiveness to external differentiation factors. We believe this is the first report demonstrating tumor suppression activity for a tetratricopeptide repeat gene family member and provides insights into the function of this family of genes in mammalian cells.

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor (M6P/IGF2R) locus predisposes patients to radiation-induced lung injury

PURPOSEnTo investigate the relationship between loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene locus and the development of radiation-induced lung injury.nnnMATERIAL AND METHODSnThirty-five lung cancer patients with both stored plasma for Transforming Growth Factor beta1 (TGFbeta1) analysis and sufficient quantities of archival pathology tissue to screen for LOH were studied. All patients had been treated with thoracic radiotherapy for their malignancy and had radiographically detectable tumor present before beginning radiotherapy. Tumor and normal cells were microdissected from archival lung cancer pathology specimens. Two polymorphisms in the 3 untranslated region of the M6P/IGF2R were used to screen for LOH. Plasma TGFbeta1 levels were measured using acid-ethanol extraction and an ELISA. TGFbeta1 and M6P/IGF2R protein expression was estimated by immunofluorescence and immunohistochemical staining. Symptomatic radiation pneumonitis was scored according to National Cancer Institute Common Toxicity Criteria without knowledge of the results of TGFbeta or LOH analyses.nnnRESULTSnOf the 35 patients, 10 were homozygous for this polymorphism (noninformative) and were excluded. Of the 25 informative patients, 13 had LOH. Twelve of 13 patients with LOH had increased pretreatment plasma TGFbeta1 levels, vs. 3/12 patients without LOH (p < 0.01). A decrease or loss of M6P/IGF2R protein in the malignant cell accompanied by increased latent TGFbeta1 protein in extracellular matrix and tumor stroma was found in tumors with LOH, suggesting that this mutation resulted in loss of function of the receptor. Seven of 13 (54%) LOH patients developed symptomatic radiation-induced lung injury vs. 1/12 (8%) of patients without LOH (p = 0.05).nnnCONCLUSIONnLoss of the M6P/IGF2R gene strongly correlates with the development of radiation pneumonitis after thoracic radiotherapy (RT). Furthermore, patients with LOH (in the setting of measurable tumor) are much more likely to have elevated plasma TGFbeta, suggesting an inability to normally process this cytokine. Thus, loss of the M6P/IGF2R gene may predispose patients to the development of radiation-induced lung injury.

Loss of heterozygosity of M6P/IGF2R gene is an early event in the development of prostate cancer

Background: The genetic events leading to initiation and/or progression of prostate cancer are not well characterized. The gene coding for the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) has recently been identified as a tumor suppressor in several types of cancer. The purpose of the present study is to determine whether the M6P/IGF2R gene is inactivated in human prostate cancer, and if so, whether this is an early or late transformational event.Methods: In total, 43 patients with prostate cancer treated by radical prostatectomy, with archival material available for analysis, were assessed for loss of heterozygosity (LOH) in the M6P/IGF2R gene using six different gene-specific nucleotide polymorphisms. Regions of tumor, normal prostate and premalignant high-grade prostate intraepithelial neoplasia (PIN) were identified and cells were excised by laser capture microdissection (LCM). DNA segments were amplified using polymerase chain reaction (PCR).Results: The M6P/IGF2R gene was polymorphic in 83.7% (36/43) of patients, and 41.7% (15/36) of these informative patients had LOH in the tumor tissue. In 11/15 patients with LOH in malignant tissue, high-grade PIN could be identified, and 63.6% (7/11) also had LOH in this premalignant tissue.Conclusions: This study is the first to find that the M6P/IGF2R gene is inactivated in prostate cancer. LOH in premalignant tissue as well suggests that mutation in the M6P/IGF2R gene is an early event in the development of prostate cancer, supporting the conclusion that it functions as a tumor suppressor gene in this disease.

IGF2R polymorphisms and risk of esophageal and gastric adenocarcinomas

The mannose‐6‐phosphate/insulin‐like growth factor 2 receptor (M6P/IGF2R) encodes a protein that plays a critical role in tumor suppression, in part by modulating bioavailability of a potent mitogen, insulin‐like growth factor‐2 (IGF2). We tested the hypothesis that the common nonsynonymous genetic variants in M6P/IGF2R c.901C > G (Leu > Val) in exon 6 and c.5002G > A (Gly > Arg) in exon 34 are associated with risk of esophageal and gastric cancers. Study participants in this population‐based study comprise 197 controls and 182 cases, including 105 with esophageal‐gastric cardia adenocarcinoma (EGA), 57 with noncardia gastric adenocarcinoma and 20 with esophageal squamous (ES) cell carcinoma. Among white males, odds ratios (ORs) were elevated in relation to carrying at least 1 c.901C > G allele for EGA [OR = 1.9; 95% confidence intervals (CIs) = 1.0–3.6] and noncardia gastric cancer (OR = 2.5; 95% CI = 1.2–5.5), but not ES. Exploratory subgroup analyses suggested that associations between EGA and this variant were stronger among irregular or nonusers of nonsteroidal anti‐inflammatory drugs (NSAIDs) (OR = 2.3; 95% CI = 1.2–4.2) and cigarette smokers (OR = 2.1; 95% CI = 1.0–4.2). An association between carrying the c.5002G > A genotype and EGA was not evident. These findings suggest that nonsynonymous polymorphisms in M6P/IGF2R may contribute to the risks of EGA and noncardia adenocarcinomas. Larger studies are required to confirm these findings.

IGF2R Genetic Variants, Circulating IGF2 Concentrations and Colon Cancer Risk in African Americans and Whites

The Mannose 6 Phosphate/Insulin-like Growth Factor Receptor-2 (IGF2R) encodes a type-1 membrane protein that modulates availability of the potent mitogen, IGF2. We evaluated the associations between IGF2R non-synonymous genetic variants (c.5002G>A, Gly1619Arg(rs629849), and c.901C>G, Leu252Val(rs8191754)), circulating IGF2 levels, and colon cancer (CC) risk among African American and White participants enrolled in the North Carolina Colon Cancer Study (NCCCS). Generalized linear models were used to compare circulating levels of IGF2 among 298 African American and 518 White controls. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association of IGF2R genetic variants and CC risk. Women homozygous for the IGF2R c.5002 G>A allele, had higher mean levels of circulating IGF2, 828 (SD=321) ng/ml compared to non-carriers, 595 (SD=217) ng/ml (p-value=0.01). This pattern was not apparent in individuals homozygous for the IGF2R c.901 C>G variant. Whites homozygous for the IGF2R c.901 C>G variant trended towards a higher risk of CC, OR=2.2 [95% CI(0.9–5.4)], whereas carrying the IGF2R c.5002 G>A variant was not associated with CC risk. Our findings support the hypothesis that being homozygous for the IGF2R c.5002 G>A modulates IGF2 circulating levels in a sex-specific manner, and while carrying the IGF2R c.901 C>G may increase cancer risk, the mechanism may not involve modulation of circulating IGF2.