Pneumatic dilation versus laparoscopic Heller myotomy for the treatment of achalasia: variables related to a good response.

Achalasia is a motor disorder characterized by esophageal aperistalsis and failure of lower esophageal sphincter relaxation. The cardinal symptoms are dysphagia, food regurgitation and weight loss. The most effective treatments are pneumatic dilation (PD) of the cardia and Heller esophageal myotomy with partial fundoplication. There is still controversy regarding which treatments should be initially done. The aims of this study were to evaluate clinical response and the variables related to good results in both treatments. Ninety-two patients with achalasia diagnosed by esophageal manometry were randomized to receive either PD or laparoscopic Heller myotomy with partial fundoplication. After the procedure, patients were followed up clinically and submitted to esophageal manometry and pH monitoring. Three months after treatment, 73% of the patients from PD group and 84% of the surgery group had good results (P = 0.19). After 2 years of follow-up, 54% of the PD group and 60% of the surgery group (P = not significant) were symptom free. Variables related to a good response to PD were a 50% drop in lower esophageal sphincter pressure (LESP) or a LESP <10 mmHg after treatment. Patients over 40 years old with LESP ≤32 mmHg before treatment and a drop in LESP >50% after treatment significantly achieved better responses after surgical treatment when compared with PD. The reflux rate was significantly higher in the PD group (27.7%) compared with the surgery group (4.7%), P = 0.003. We concluded that surgical treatment and PD for achalasia are equally effective even after 2 years of follow-up. The choice of treatment for achalasia should be based on the following parameters: treatment availability, rate of good results, complication rates, variables related to good responses and also the patient's wish.

Global methylation analysis identifies PITX2 as an upstream regulator of the androgen receptor and IGF-I receptor genes in prostate cancer.

The insulin-like growth factor-I (IGF-IR) and androgen (AR) receptors are important players in prostate cancer. Functional interactions between the IGF-I and androgen signaling pathways have crucial roles in the progression of prostate cancer from early to advanced stages. DNA methylation is a major epigenetic alteration affecting gene expression. Hypermethylation of tumor suppressor promoters is a frequent event in human cancer, leading to inactivation and repression of specific genes. The aim of the present study was to identify the entire set of methylated genes ("methylome") in a cellular model that replicates prostate cancer progression. The methylation profiles of the P69 (early stage, benign) and M12 (advanced stage, metastatic) prostate cancer cell lines were established by treating cells with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza) followed by DNA microarray analysis. Comparative genome-wide methylation analyses of 5-Aza-treated versus untreated cells identified 297 genes overexpressed in P69 and 191 genes overexpressed in M12 cells. 102 genes were upregulated in both benign and metastatic cell lines. In addition, our analyses identified the PITX2 gene as a master regulator upstream of the AR and IGF-IR genes. The PITX2 promoter was semi-methylated in P69 cells but fully methylated (i. e., silenced) in M12 cells. Epigenetic regulation of PITX2 during the course of the disease may lead to orchestrated control of the AR and IGF signaling pathways. In summary, our results provide new insights into the epigenetic changes associated with progression of prostate cancer from an organ confined, androgen-sensitive disorder to an aggressive, androgen-insensitive disease.

Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism.

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycin-induced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

Activation of an imprinted allele of the insulin-like growth factor II gene implicated in rhabdomyosarcoma.

The insulin-like growth factor II (IGF2) gene is exclusively silent at the maternal allele in the mouse as well as in normal human tissues and is expressed at a high level in rhabdomyosarcoma (RMS). We report here that the normally imprinted allele of the IGF2 gene is activated in RMS tumors as well as in one RMS cell line. Since overexpression of IGF2 has been shown to be important in the pathogenesis of RMS, our data suggest that loss of imprinting (LOI) may lead to overexpression of IGF2 and play an important role in the onset of RMS. Furthermore, embryonal RMS usually has loss of heterozygosity (LOH) with paternal disomy of the IGF2 locus. One informative embryonal RMS tumor evaluated in this study was heterozygous at the IGF2 allele and had LOI, raising the possibility that LOI may be the functional equivalent of LOH in this tumor with both events leading to overexpression of IGF2.

Secretory protein traffic. Chromogranin A contains a dominant targeting signal for the regulated pathway.

Secretory proteins are targeted into either constitutive (secreted upon synthesis) or regulated (stored in vesicles and released in response to a secretagogue) pathways. To investigate mechanisms of protein targeting into catecholamine storage vesicles (CSV), we stably expressed human chromogranin A (CgA), the major soluble protein in human CSV, in the rat pheochromocytoma PC-12 cell line. Chromaffin cell secretagogues (0.1 mM nicotinic cholinergic agonist, 55 mM K+, or 2 mM Ba++) caused cosecretion of human CgA and catecholamines from human CgA-expressing cells. Sucrose gradients colocalized human CgA and catecholamines to subcellular particles of the same buoyant density. Chimeric proteins, in which human CgA (either full-length [457 amino acids] or truncated [amino-terminal 226 amino acids]) was fused in-frame to the ordinarily nonsecreted protein chloramphenicol acetyltransferase (CAT), were expressed transiently in PC-12 cells. Both constructs directed CAT activity into regulated secretory vesicles, as judged by secretagogue-stimulated release. These data demonstrate that human CgA expressed in PC-12 cells is targeted to regulated secretory vesicles. In addition, human CgA can divert an ordinarily non-secreted protein into the regulated secretory pathway, consistent with the operation of a dominant targeting signal for the regulated pathway within the peptide sequence of CgA.

Insulin-like growth factor II-mediated proliferation of human neuroblastoma.

Neuroblastoma is an embryonal tumor that typically arises in cells of the developing adrenal medulla. IGF-II mRNA is expressed at high levels in the adrenal cortex before birth but it is not detectable until after birth in the adrenal medulla. Neuroblastoma cell lines corresponding to early adrenal medullary precursors did not express IGF-II, although all three cell lines we tested were growth stimulated by IGF-II. Cell lines corresponding to more mature adrenal medullary cells expressed IGF-II, and one, SK-N-AS, grows by an IGF-II autocrine mechanism (J. Clin. Invest. 84:829-839) El-Badry, Romanus, Helman, Cooper, Rechler, and Israel. 1989. An examination of human neuroblastoma tumor tissues for IGF-II gene expression using in situ hybridization histochemistry revealed that IGF-II is expressed by tumor cells in only 5 of 21 neuroblastomas, but is detectable in cells of nonmalignant tissues including adrenal cortical cells, stromal fibroblasts, and eosinophils in all 21 tumors. These findings indicate that IGF-II may function as an autocrine growth factor for some neuroblastomas and as a paracrine growth factor for others. They suggest that the growth regulatory pathways utilized by neuroblastoma mimic those used in the precursor cell type from which individual tumors arise.

Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) mRNA was increased in two of eight neuroblastomas and in eight of eight pheochromocytomas, tumors of the adrenal medulla that occur in childhood and adulthood, respectively. RNA encoding the type I IGF receptor, the receptor thought to mediate the mitogenic effects of IGF-I and IGF-II, also was uniformly expressed in these cells. To assess the role of IGF-II in the growth of these tumor cells, we have used the SK-N-AS cultured neuroblastoma cell line, which can be continuously propagated in mitogen-free medium, as a model system. Our results strongly suggest that IGF-II, synthesized by SK-N-AS cells and acting through type I IGF receptors, contributes to the autonomous growth of this tumor cell line. (a) SK-N-AS cells synthesized large amounts of IGF-II RNA and secreted greater than 50 ng/ml of IGF-II (as determined by specific radioimmuno- and radioreceptor assays). Little, if any, IGF-I RNA or immunoreactive IGF-I were detected. (b) SK-N-AS cells possess type I IGF receptors. (c) Exogenous IGF-I and IGF-II stimulated DNA synthesis in SK-N-AS cells, and this stimulation was abolished by a blocking antibody to the type I IGF receptor. (d) This anti-receptor antibody also abolished the multiplication of SK-N-AS cells in the absence of added mitogens. We conclude that IGF-II is an autocrine growth factor for SK-N-AS cells and suggest that this mechanism may contribute to the growth of some adrenal medullary tumors.

Chromogranin A expression in normal and malignant human tissues.

We used a recombinant cDNA probe for human chromogranin A to measure the expression of mRNA encoded by this gene in a variety of normal human tissues and tumor specimens using Northern blot and in situ hybridization analysis. With few exceptions, the expression of chromogranin A mRNA appears to be restricted to normal tissues and tumors of neuroendocrine lineage. However, we have detected mRNA expression of this gene in 1 of 14 cell lines and 2 of 13 tumor specimens of colon adenocarcinoma. The finding of chromogranin A expression in some colon carcinomas suggests that a previously unrecognized subgroup of these tumors has neuroendocrine features. The detection of this subgroup demonstrates the potential for improving tumor classification through the use of techniques and reagents developed by recombinant DNA technology.

All-trans-retinoic acid inhibits the growth of human rhabdomyosarcoma cell lines.

We have been evaluating the role of all-trans-retinoic acid (RA) in the differentiation and growth of human rhabdomyosarcoma (RMS) cell lines. Treatment of both embryonal (RD) and alveolar (RH30) human RMS cell lines with all-trans-RA resulted in a dose-dependent inhibition of cell growth with a maximal inhibition of 92 and 66%, respectively, at 5 x 10(-6) M. When 13-cis-RA was used under identical experimental conditions, maximal growth inhibition was 41 and 37%, respectively. This stereo-specific growth inhibition was not associated with morphological or biochemical evidence of myogenic differentiation. Furthermore, all-trans-RA demonstrated no evidence of competition with binding of insulin-like growth factor II (IGF-II), an autocrine growth factor in RMS, to its membrane receptor as evaluated by an [125I]IGF-I-receptor-binding assay. Attempts to rescue all-trans-RA growth-inhibited RMS cells with exogenous IGF-II resulted in no increase in growth compared to cells treated with all-trans-RA alone. We conclude that RA inhibits the growth of human RMS cell lines in a dose-dependent, stereo-specific manner, is not associated with differentiation, and does not appear to be directly related to IGF-II.

Suramin inhibits the growth of human rhabdomyosarcoma by interrupting the insulin-like growth factor II autocrine growth loop.

Suramin is a polysulfonated naphthyl-urea with antineoplastic activity that binds various peptide growth factors. Since we previously demonstrated that insulin-like growth factor II (IGF-II) is an autocrine growth factor in human rhabdomyosarcoma (RMS), we studied the effect of suramin on the growth of human RMS cells. Suramin caused a dose-dependent decrease of RMS cell number grown either in 10% fetal bovine serum or in serum-free medium (half-maximal effective dose in mitogenic assays, 1.6 x 10(-4) and 9 x 10(-5) M, respectively). IGF-II and IGF-I added to RMS cells in the presence of suramin reversed the suramin-induced inhibition of cell growth. Since IGF-II exerts its mitogenic effects on RMS cells by binding to the type I receptor, we performed radioreceptor assays using 125I-IGF-I and found that suramin displaced 125I-IGF-I from the type I IGF receptor. There was an excellent correlation between the doses of suramin effective in inhibiting the growth of RMS cells and those that displaced the binding of IGF-I. Our data indicate that suramin exerts its effect on RMS cell growth by interfering with the binding of IGF-II to the type I IGF receptor, thereby interrupting the IGF-II autocrine growth in these cells. Disrupting autonomous growth of RMS may be a promising novel therapeutic approach.

In vivo treatment with antibody against IGF-1 receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34cdc2.

In a previous study, we have shown that insulin-like growth factor type 2 (IGF-2) functions as an autocrine growth factor in human rhabdomyosarcoma (RMS) cell lines. In addition, we demonstrated that the inhibition of binding of IGF-2 to the IGF-1 receptor, mediated by suramin, blocked the growth of RMS cells in vitro. We now report that, in vivo, a specific IGF-1 receptor blocking antibody (alpha IR-3), but not suramin, suppresses RMS tumor growth. Both progression of tumor growth in tumor-bearing animals and formation of newly established tumors were suppressed by treatment with alpha IR-3. Histological analysis of tumors from treated animals did not reveal necrotic lesions, implying that the treatments had no cytotoxic effect. The decrease in tumor growth was associated with a decrease of p34cdc2, a cellular protein involved in cell cycle regulation, suggesting that treatment resulted in the arrest of cellular proliferation. We speculate, therefore, that agents which block the IGF signaling pathway may find application in treatment of RMS.

Cytotoxic activity of a recombinant fusion protein between insulin-like growth factor I and Pseudomonas exotoxin.

A chimeric toxin in which the cell binding domain of Pseudomonas exotoxin was replaced with mature human insulin-like growth factor I (IGF-I) was produced in Escherichia coli. This protein, IGF-I-PE40, was cytotoxic to human cell lines derived from a variety of tumor types, with a breast carcinoma line (MCF-7) and two hepatoma lines (HEP3B and HEPG2) showing the highest sensitivity to the toxin. The specificity of IGF-I-PE40 cytotoxicity was confirmed through competition with excess IGF-I and through blockage of toxin binding using an antibody specific to the type I IGF receptor. A potential interaction between the toxin and soluble IGF-binding proteins was also demonstrated. IGF-I-PE40 may be useful in the selective elimination of cells bearing the type I IGF receptor.

Human osteosarcoma cell lines are dependent on insulin-like growth factor I for in vitro growth.

Osteogenic sarcoma is the most common bone tumor of childhood and typically occurs during the adolescent growth spurt when growth hormone and insulin-like growth factor I (IGF-I) may be at their lifetime highest levels. Since IGF-I is involved in normal bone growth and differentiation, we have evaluated the possible role of IGF-I signaling in the growth of human osteogenic sarcoma cell lines. In this study, we demonstrate that in vitro survival of cells is dependent on exogenously supplied IGF-I. Furthermore, we show that these cells display functional IGF-I receptors on their surface and that in vitro growth is inhibited by blocking these receptors either by monoclonal antibodies or by antisense oligonucleotides. These data demonstrate that human osteogenic sarcoma cell lines are dependent on signaling through the IGF-I receptor for in vitro survival and proliferation. Furthermore, they suggest that modulation of the growth hormone/IGF-I axis may affect the growth of these tumors in vivo.

Insulin-like growth factor II and PAX3-FKHR cooperate in the oncogenesis of rhabdomyosarcoma.

The mouse myoblast C2C12 cell line transfected singly with cDNA for Pax-3, PAX3-FKHR, or insulin-like growth factor (IGF) II or cotransfected with IGF-II plus Pax-3 or with IGF-II plus PAX3-FKHR genes showed an altered morphology, a lack of differentiation, and higher proliferation rates in vitro. On s.c. injection into nude mice, tumors grew from transfected cell lines but not from cells transfected with the empty vector. Tumors derived from IGF-II/PAX3-FKHR- and IGF-II-transfected cells grew most rapidly. Cotransfection of IGF-II plus Pax-3 induced tumors comprised highly differentiated striated muscle cells; Pax-3, PAX3-FKHR, or IGF-II transfection produced tumors at varying stages of differentiation. Tumors derived from IGF-II plus PAX3-FKHR-cotransfected cells were composed of undifferentiated cells. This was the only tumor type to infiltrate the underlying muscle. The most angiogenesis and the least apoptosis were observed in the latter tumors. These results support the hypothesis that PAX3-FKHR interacts with IGF-II to play a critical role in the oncogenesis of rhabdomyosarcoma.

XAGE-1, a new gene that is frequently expressed in Ewing's sarcoma.

Our previous expressed sequence tag database analysis indicates that XAGE-1 is frequently found in Ewing's sarcoma and alveolar rhabdomyosarcoma (U. Brinkmann et al., Cancer Res., 59: 1445-1448, 1999). Using Northern blots and RNA dot blots, we have now found that XAGE-1 is highly expressed in normal testis, in seven of eight Ewing's cell lines, in four of nine Ewing's sarcoma patient samples, and in one of one alveolar rhabdomyosarcoma patient sample. The gene is located on the X chromosome. The full-length cDNA contains 611 bp and predicts a protein of Mr 16,300 with a potential transmembrane domain at the NH2 terminus. XAGE-1 shares homology with GAGE/PAGE proteins in the COOH-terminal end. These findings could be valuable for cancer diagnosis and cancer immunotherapy.

Neuropeptide Y expression in the developing adrenal gland and in childhood neuroblastoma tumors.

Neuropeptide Y (NPY) expression is limited to tissues of the central and peripheral nervous system. In the adrenal gland, NPY is found in a subset of cells of the adrenal medulla. Using in situ hybridization analysis, NPY mRNA expression was characterized during human fetal adrenal medullary development. We found a biphasic pattern of NPY mRNA expression during the development of the human adrenal medulla. NPY mRNA is detectable at the earliest evaluable time point (7.5 weeks of gestational age) through 18 weeks of gestational age, and is then not detectable until 8 months after birth. We also analyzed NPY mRNA expression in neuroblastoma tumors, which often arise in the adrenal medulla. Thirty-eight neuroblastoma tumors were analyzed for NPY mRNA expression using in situ hybridization. We found NPY mRNA expression in 30 of 38 tumors; 15 of 15 Stage IVS tumors from children under 1 year of age at diagnosis expressed NPY mRNA, whereas 0 of 4 Stage IV tumors from children less than 1 year of age at diagnosis expressed NPY mRNA. These data suggest that in children under 1 year of age at diagnosis, Stage IVS and Stage IV neuroblastoma may be marked by the presence or absence, respectively, of NPY mRNA expression. Moreover, since NPY is expressed for only a short period of time during embryogenesis, these tumors may arise from different neuroblast populations occurring during the course of adrenal medullary development.

Metastasis-associated differences in gene expression in a murine model of osteosarcoma.

Despite advances in the management of osteosarcoma (OSA) and other solid tumors, the development of metastasis continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastasis, we have applied cDNA microarrays to a recently described murine model of OSA that is characterized by orthotopic tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and cell line variants that differ in metastatic potential. Microarray analysis defined 53 genes (of 3166 unique cDNAs) that were differentially expressed between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) OSA models. By review of the literature, these differentially expressed genes were assigned to six nonmutually exclusive metastasis-associated categories (proliferation and apoptosis, motility and cytoskeleton, invasion, immune surveillance, adherence, and angiogenesis). Functional studies to evaluate K7M2 and K12 for differences in each of these metastasis-associated processes revealed enhanced motility, adherence, and angiogenesis in the more aggressive K7M2 model. For this reason, 10 of the 53 differentially expressed genes that were assigned to the motility and cytoskeleton, adherence, and angiogenesis categories were considered as most likely to define differences in the metastatic behavior of the two models. Ezrin, a gene not described previously in OSA, with functions in motility, invasion, and adherence, was 3-fold overexpressed in K7M2 compared with K12 by microarray. Differential expression for RNA was confirmed by Northern analysis and for protein by immunostaining. Alterations in ezrin protein levels and concomitant cytoskeletal changes in our model confirmed predictions from the arrays. The potential relevance of ezrin in OSA was suggested by its expression in five of five human OSA cell lines. This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis.

Regulation of insulin-like growth factor II P3 promotor by p53: a potential mechanism for tumorigenesis.

Human insulin-like growth factor (IGF)-II mRNA has been shown to be expressed at high levels in a variety of tumors, including rhabdomyosarcomas. In addition, many tumors have alterations in p53 expression. To investigate whether p53 regulates IGF-II gene expression, we transfected wild-type p53 expression vectors and luciferase constructs driven by IGF-II P3 promotors into multiple cell lines. We found that p53 reduced, in a dose-dependent manner, both endogenous IGF-II P3 transcripts and transfected P3 luciferase expression. The inhibition of P3 luciferase expression by p53 was more pronounced in the two cell lines that expressed mutant p53 protein, RD, and HTB114. The element responsible for this inhibition was mapped to the minimal promoter region. We also transfected an HPV-16 E6 expression plasmid into CCL13 cells containing functional p53 and found that E6 up-regulated IGF-II P3 activity. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein to the TATA motif of P3, although both could directly associate with human TATA-binding protein. Our results suggest that p53 may play a role in regulation of IGF-II gene expression.

Expression of neuroendocrine cell markers L-dopa decarboxylase, chromogranin A, and dense core granules in human tumors of endocrine and nonendocrine origin.

We evaluated the usefulness of L-dopa decarboxylase (DDC) as a tumor marker of neuroendocrine (NE) cell differentiation by measuring its expression in 432 human tumors of diverse types and origins. A subset of these tumors and cell lines derived from them also were studied for expression of two other general NE cell markers, chromogranin A (CgA) and dense core granules (DCG). High concentrations of DDC were present in 96 of 117 (82%) tumors recognized to be of NE or neural origin. As expected, endocrine tumors not recognized to be of NE cell origin, as well as leukemias, lymphomas, sarcomas, melanomas, and germ cell tumors, lacked DDC expression. Of interest, modest concentrations of DDC were present in 46 of 220 (21%) nonendocrine carcinomas, especially non-small cell lung and colorectal carcinomas. We studied concordant expression of the three NE cell markers in lung and colorectal tumors and cell lines. In both tumor types there was nearly 100% concordance between CgA and DCG expression. There was an excellent correlation between DDC and CgA expression in lung cancers, both small cell and non-small cell, but DDC positive colorectal carcinomas usually lacked CgA expression. We conclude: (a) DDC is an excellent cellular marker for tumors of the NE cell system; (b) about 20% of carcinomas not of NE cell origin, especially non-small cell lung and colorectal carcinomas, express DDC, suggesting a common endodermal origin of all of the respiratory and gastrointestinal mucosal cells; and (c) CgA and DCG are expressed concordantly, indicating that CgA expression may be used as a substitute for ultrastructural examination of tumors for DCG expression.