Downregulation of IGFBP2 is associated with resistance to IGF1R therapy in rhabdomyosarcoma.

Agents targeting the insulin-like growth factor-1 receptor (IGF1R) are in clinical development, but, despite some initial success of single agents in sarcoma, response rates are low with brief durations. Thus, it is important to identify markers predictive of response, to understand mechanisms of resistance, and to explore combination therapies. In this study, we found that, although associated with PAX3-FKHR translocation, increased IGF1R level is an independent prognostic marker for worse overall survival, particularly in patients with PAX3-FKHR-positive rhabdomyosarcoma (RMS). IGF1R antibody-resistant RMS cells were generated using an in vivo model. Expression analysis indicated that IGFBP2 is both the most affected gene in the insulin-like growth factor (IGF) signaling pathway and the most significantly downregulated gene in the resistant lines, indicating that there is a strong selection to repress IGFBP2 expression in tumor cells resistant to IGF1R antibody. IGFBP2 is inhibitory to IGF1R phosphorylation and its signaling. Similar to antibodies to IGF1/2 or IGF2, the addition of exogenous IGFBP2 potentiates the activity of IGF1R antibody against the RMS cells, and it reverses the resistance to IGF1R antibody. In contrast to IGF1R, lower expression of IGFBP2 is associated with poorer overall survival, consistent with its inhibitory activity found in this study. Finally, blocking downstream Protein kinase B (AKT) activation with Phosphatidylinositide 3-kinases (PI3K)- or mammalian target of rapamycin (mTOR)-specific inhibitors significantly sensitized the resistant cells to the IGF1R antibody. These findings show that constitutive IGFBP2 downregulation may represent a novel mechanism for acquired resistance to IGF1R therapeutic antibody in vivo and suggest various drug combinations to enhance antibody activity and to overcome resistance.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

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.

Insulin-like growth factor 1 receptor antibody induces rhabdomyosarcoma cell death via a process involving AKT and Bcl-x(L).

Insulin-like growth factors (IGFs) and their receptor, IGF-1 receptor (IGF1R), have important roles in growth, development, stress response, aging and cancer. There are many agents that inhibit IGF1R in oncology clinical development, and in some cases, they have been associated with rapid tumor regression. However, it is not clear by which process these targeted agents induce cancer cell death and how to predict such tumor responses. Here, we showed that IGF1R antibody led to rapid cell death and tumor regression in some rhabdomyosarcoma (RMS) cells. Mechanistic analysis revealed a rapid onset of mitochondrial-dependent apoptosis, including mitochondrial depolarization, cytochrome C release and the activation of specific caspases. The antibody sensitive cells had greater dependence on AKT for maintaining downstream signaling and the expression of a constitutively active AKT, which restored AKT-signaling in these cells, inhibited anti-IGF1R induced cell death. Further analysis showed IGF1R antibody-induced hypophosphorylation of BAD and activation of downstream BAX. Interestingly, the examination of RMS cell lines and tumors revealed an inverse correlation between elevated IGF1R and Bcl-2 level (P=0.033), with the sensitive cells lacking Bcl-2 expression. The overexpression of BAD specific target, Bcl-x(L), conferred resistance, whereas Bcl-x(L) knockdown sensitized cells lacking Bcl-2 to anti-IGF1R-induced cell death. We propose that RMS pathogenesis involves increased IGF1R expression that enhances AKT and Bcl-x(L)-mediated cell survival, and the blockage of IGF1R results in inhibition of survival signal from Bcl-x(L) and cell death in the sensitive Bcl-2 negative cells.

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.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy. It was hypothesized that the fusion breakpoint sequences associated with SS, CCS, and DSRCT can serve as tumor-specific neoantigens. To test this, peptides corresponding to the fusion breakpoints were designed and assessed for ability to bind to various class I HLA molecules. Two peptides derived from the SS breakpoint specifically bind the HLA-B7 antigen, and a 10-amino acid minimal epitope was identified for this interaction. Specific binding of a SS peptide and a CCS peptide to HLA-B27 molecule was also observed. Finally, a peptide designed from the DSRCT breakpoint specifically binds the HLA-A3 molecule, and a 9-amino acid optimal epitope was identified for this interaction. The physiological/immunological relevance of these peptide/MHC interactions was demonstrated by the induction of SS-specific CTLs from normal donor lymphocytes using in vitro stimulation with autologous, peptide-pulsed dendritic cells and by the ability of these CTLs to lyse human SS tumor cells endogenously expressing the full-length fusion protein. These results suggest that sequences in the fusion region of sarcoma-associated chimeras can bind class I HLA molecules and serve as neoantigens. These may be useful for the development of novel immunotherapies for sarcoma patients with appropriate HLA molecules and tumors bearing these translocations.

Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: quantitation by real-time polymerase chain reaction.

PURPOSE: A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens. PATIENTS AND METHODS: Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays. RESULTS: MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR. CONCLUSION: These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.

Targeting pediatric malignancies for T cell-mediated immune responses.

Successful immune targeting of malignancies hinges upon the ability to activate specific T-cell populations to recognize and attack tumor but spare normal vital tissues. Investigators in the field of tumor immunology are currently utilizing at least three distinct approaches toward this goal. In the first approach, molecular targets of cytolytic T cells which spontaneously develop in tumor-bearing patients have been identified and are subsequently used as immunogens in immunotherapy trials. Whereas this approach originally focused upon the identification of tumor antigens in the immune-responsive tumors malignant melanoma and renal cell carcinoma, it surprisingly led to the identification of a variety of molecules that are now known to be expressed in other common pediatric and adult tumors. In the second approach, tumor-specific molecules (eg, mutant p53 and chromosomal translocations) that have been identified in individual tumors during the study of neoplastic transformation are used as immunogens. Because chromosomal translocations are common in pediatric tumors, such targets may be of particular interest in pediatric oncology. In the third approach, immunization with whole tumor cell components is undertaken with the assumption that the most immunogenic molecules within the tumor will dominate the immune response induced. The benefits and limitations for each approach, particularly as it pertains to the development of immunotherapy for pediatric tumors, are discussed in this article.

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.

Targeting tumor specific translocations in sarcomas in pediatric patients for immunotherapy.

In an effort to develop more effective therapies for various sarcomas in pediatric patients, the authors have focused on using recurrent tumor-specific translocations as potential novel tumor antigens. In general, these translocations generate fusion transcription factors. Because cytotoxic T cell lymphocyte receptors recognize peptide fragments bound to major histocompatibility complex Class 1 molecules, it is possible that unique peptides spanning the translocation breakpoint region may be processed, bound to major histocompatibility complex Class I molecules and displayed on the tumor cell surface where they could be susceptible to cytotoxic T cell lymphocyte killing. The authors have investigated the PAX-3-FKHR fusion product seen in alveolar rhabdomyosarcoma, and the EWS-FLI-1 fusion product seen in Ewing's sarcoma. Peptides spanning these fusion regions contain potential major histocompatibility complex Class 1 and Class II binding motifs suggesting they may serve as novel T cell antigens. Preliminary mouse experiments suggest that cytotoxic T cell lymphocytes specific for the PAX-3-FKHR fusion peptide can be generated and can recognize and kill tumor cells bearing the PAX-3-FKHR fusion protein. Clinical trials are ongoing to determine whether this approach will be useful.

Inguinal hernia in patients with Ewing sarcoma: a clue to etiology.

BACKGROUND: Various congenital anomalies have been associated with childhood cancer, but as yet no anomaly has been consistently found with Ewing sarcoma (ES). Recently a large case-control study of ES patients reported a greater number of hernias in both cases and their sibling controls than in population controls. Most of these hernias were inguinal. Because these anomalies were also reported previously in two case series, we looked for inguinal hernias in a different population of ES patients. PROCEDURE: We abstracted medical records for 306 pathologically confirmed ES/primitive neuroectodermal tumor (PNET) patients seen at NIH between 1960 and 1992. Epidemiological data on demographics and medical conditions were analyzed. The frequency of anomalies was compared to expected rates to calculate relative risk and confidence intervals. RESULTS: Anomalies were present in 67 (22%) cases. A particular anomaly, inguinal hernia, was reported for 13 (5%) NIH cases. Compared to population estimates for white children, the relative risk of inguinal hernia among white NIH cases was 13.3 (95% CI 3.60-34.1) for females and 6.67 (95% CI 2.67-13.7) for males. CONCLUSIONS: The findings of inguinal hernias in some patients with ES suggest that a disruption in normal embryological development occurred. This may provide an important clue to the etiology of ES. We hypothesize that these hernias may relate to an in utero exposure or indicate an underlying genetic disorder. Future studies should carefully evaluate ES families for genetic disease and explore environmental factors. Med. Pediatr. Oncol. 34:195-199, 2000. Published 2000 Wiley-Liss, Inc.

Autocrine Transforming Growth Factor-beta Growth Pathway in Murine Osteosarcoma Cell Lines Associated with Inability to Affect Phosphorylation of Retinoblastoma Protein.

Purpose. Production of active transforming growth factor-beta (TGF-beta ) by human osteosarcoma may contribute to malignant progression through mechanisms that include induction of angiogenesis, immune suppression and autocrine growth stimulation of tumor cell growth.To study events associated with induction of cell proliferation by TGF-beta , we have evaluated the TGF-beta pathway in two murine osteosarcoma cell lines, K7 and K12.Results. Northern and immunohistochemical analyses show that each cell line expressesTGF-beta1 and TGF-beta3 mRNA and protein. Both cell lines secrete activeTGF-beta 1 and display a 30-50% reduction in growth when cultured in the presence of a TGF-beta blocking antibody. Expression of TGF-beta receptors TbetaRI, TbetaRII and TbetaRIII is demonstrated by affinity labeling with (125) -TGF-beta 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed. Smads 2 and 3 are phosphorylated in response toTGF-beta , while pRb phosphorylation in each osteosarcoma cell line is not affected by either exogenousTGF-beta or TGF-beta antibody.Conclusions. The data implicate events downstream of Smad activation, including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-beta , and indicate that this murine model of osteosarcoma is valid for investigating the roles of autocrineTGF-beta in vivo.

Rhabdomyosarcoma--working out the pathways.

Rhabdomyosarcomas constitute a collection of childhood malignancies thought to arise as a consequence of regulatory disruption of skeletal muscle progenitor cell growth and differentiation. Our understanding of the pathogenesis of this neoplasm has recently benefited from the study of normal and malignant myogenic cells in vitro, facilitating the identification of diagnostic cytogenetic markers and the elucidation of mechanisms by which myogenesis is regulated. It is now appreciated that the delicate balance between proliferation and differentiation, mutually exclusive yet intimately associated processes, is normally controlled in large part through the action of a multitude of growth factors, whose signals are interpreted by members of the MyoD family of helix - loop - helix proteins, and key regulatory cell cycle factors. The latter have proven to be frequent targets of mutational events that subvert myogenesis and promote the development of rhabdomyosarcoma. Although significant progress has been made in the treatment of rhabdomyosarcoma, patients presenting with metastatic disease or certain high risk features are still faced with a dismal prognosis. Only now are genetically engineered mouse models becoming available that are certain to provide fresh insights into the molecular/genetic pathways by which rhabdomyosarcomas arise and progress, and to suggest novel avenues of therapeutic opportunity.

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression.

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.

AP-2 may contribute to IGF-II overexpression in rhabdomyosarcoma.

The human insulin-like growth factor II gene is regulated in a development-dependent manner and is not expressed in most adult tissues. However, high levels of insulin-like growth factor II mRNA are detected in many human tumors including rhabdomyosarcoma, an embryonal tumor of skeletal muscle origin. In this study, we demonstrate that the developmentally regulated transcription factor AP-2 is expressed at higher levels in human fetal skeletal muscle and rhabdomyosarcoma cells compared to human adult skeletal muscle. Endogenous insulin-like growth factor II mRNA derived from the P3 as well as transfected P3 promoter activity were modestly and consistently increased to the same extent following treatment of the rhabdomyosarcoma cell line RD with forskolin, a compound implicated in AP-2 transactivation. This effect of AP-2 on increased transcriptional activity was confirmed by nuclear run-on assays. Expression of AP-2B, a dominant-negative inhibitor of AP-2, suppressed the P3 promoter activity in AP-2 expressing RD cells. Furthermore, five AP-2 protected regions corresponding to six AP-2 specific binding sites were detected in the insulin-like growth factor II P3 promoter. These data together suggest that AP-2 may contribute to the high expression of IGF-II in rhabdomyosarcoma cells.

Biallelic expression of all four IGF-II promoters and its association with increased methylation of H19 gene in human brain.

The human IGF-II gene is maternally imprinted in all tissues except adult liver and the choroid plexus/leptomeninges of the central nervous system where IGF-II is biallelically expressed. In human liver, it has recently been reported that this biallelic expression only involves the promoter P1 while the promoters P2-P4 direct IGF-II transcription monoallelically. To explore whether or not biallelic expression of the IGF-II promoters in human CNS displays the same pattern as in liver, we examined the allelic expression status of the four IGF-II promoters in human brain. We found that all four IGF-II promoters in human fetal and adult brain were expressed from both parental alleles. Furthermore, the levels of methylation of the 3' region of H19 gene in fetal brain were higher than those in other tissues with monoallelic expression of IGF-II. Since similar findings have been reported in Wilms' tumor, these data suggest a similar mechanism may be responsible for loss of imprinting of IGF-II in normal brain and Wilms' tumor.

Expression of a kinase-deficient IGF-I-R suppresses tumorigenicity of rhabdomyosarcoma cells constitutively expressing a wild type IGF-I-R.

Previous results have shown that the insulin-like growth factor type I receptor (IGF-I-R) plays a critical role in the control of rhabdomyosarcoma (RMS) growth. The purpose of this study was to investigate whether a mutated IGF-I-R, when expressed in RMS cells, may interfere with the function of the endogenous wild-type IGF-I-R. We also examined whether the expression of a mutated IGF-I-R may induce phenotypic changes in RMS cells. We used here the mutated IGF-I-R with a lysine to arginine residue 1003 substitution, called IGF-I-KR, which carries a mutation in the ATP-binding domain of the intracellular beta subunit, while the extracellular, ligand binding alpha subunit remains unchanged. We observed that the expression of this mutated IGF-I-KR markedly decreased the response of RMS cells to stimulation with IGF-I. While stimulation with IGF-I increases the autophosphorylation of IGF-I-R in the parent cells, stimulation with IGF-I failed to produce a comparable increase in autophosphorylation in the cells expressing the mutated IGF-I-KR. We also observed a decreased plating efficiency of cells expressing the mutated IGF-I-KR. Consistently, a decrease of RMS growth in vivo was observed in an animal model. Our data suggest that the IGF/IGF-I-R signaling pathway may be inhibited by expressing a mutated IGF-I-KR and that such a mutant gene could be utilized in developing novel therapeutic strategies to suppress RMS growth. 1998.

p53 regulates human insulin-like growth factor II gene expression through active P4 promoter in rhabdomyosarcoma cells.

The developmentally regulated human insulin-like growth factor II (IGFII) gene is expressed at high levels in many types of tumors and promotes the proliferation of tumor cells with a high incidence of p53 gene defects. We have previously shown that p53 inhibits IGFII P3 promoter activity and decreases endogenous IGFII gene expression derived from the P3 promoter in rhabdomyosarcomas by interfering with TBP binding to the TATA element of the IGFII P3 promoter. In this report, we demonstrate that wild-type p53 expression in rhabdomyosarcoma cell lines containing mutant p53 leads to a decrease in the activity of another active IGFII promoter, P4, and a 5-fold reduction of IGFII mRNA derived from the P4 promoter. This inhibition of P4 activity is associated with direct binding of p53 to the P4 proximal promoter element despite the lack of a p53 consensus binding site. Our results suggest that p53 inhibits IGFII P4 promoter activity by a mechanism different than its effect on the P3 promoter. These data also supply further evidence of cross-talk between the IGF and p53 signaling pathways.