IGF-IR Antibody Research Articles

Abstract P6-12-05: Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells

Abstract Insulin receptor substrate (IRS) proteins are adaptor proteins downstream of insulin-like growth factor (IGF) and insulin receptors. IRS proteins play a role in cancer biology, with IRS1 function linked to mitogenesis and survival and IRS2 associated with metastasis. Moreover, this family of adaptor proteins is also involved in the signal transduction of many other transmembrane receptors. Therefore IRS proteins could be potential therapeutic targets for cancer therapy. To test the function of IRS-1, we created a doxycycline inducible IRS1 shRNA and stably transfected MCF-7L breast cancer cells. The level of IRS1 protein knockdown varied within different clones from 50 to 95% reduction compared to non-induced cells. IRS2 mRNA and protein levels were not significantly affected. We chose a high (3G5) and intermediate (3A7) IRS1 knockdown clones for further study. Doxycycline treated 3G5 and 3A7, showed partial inhibition of IGF-I, IGF-II and insulin stimulated phosphorylation of IRS1(Tyr1222) and AKT. pErk1,2 were slightly increased and pIGF-IR was unchanged compared to doxycycline non-treated cells. IRS-2 tyrosine phosphorylation was stimulated when IRS-1 was suppressed and may account for the persistent downstream cell signaling. Anti IGFIR antibody dalotuzumab (20µg/ml) effectively inhibited cell signaling stimulated by IGF-I, IGF-II and insulin in both doxycycline treated and non-treated cells. There was no obvious difference in cell signaling pattern between plus and minus doxycycline treated cells within 24 hour time course. In monolayer cell growth assays, IGF-I, IGF-II, insulin and estradiol growth stimulation were significantly inhibited when IRS-1 was suppressed in 3G5 cells. In 3A7 cells, which have a lower level of IRS-1 suppression, growth stimulation was moderately inhibited. In anchorage independent growth assays stimulated by IGF-I, colony number and size were inhibited by doxycycline-induced IRS-1 suppression with a reduction of 80% in 3G5 cells and 50% reduction in 3A7 cells. These results show that growth of MCF-7L cells are dependent on IRS-1 expression. Reduced IRS1 can hinder cancer cell growth and tumorigenicity even when signaling was not impaired potentially due to compensation by other adaptor proteins. We conclude that suppression of IRS-1 may inhibit several growth regulatory pathways in breast cancer and could serve as a potential drug target. Citation Format: Xihong Zhang, Sidhant Varma, Douglas Yee. Inducible suppression of insulin receptor substrate I inhibits IGF-I/insulin/estradiol dependent cell growth in MCF-7L breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-05.

IGF-1R Inhibition Activates a YES/SFK Bypass Resistance Pathway: Rational Basis for Co-Targeting IGF-1R and Yes/SFK Kinase in Rhabdomyosarcoma

The insulin-like growth factor 1 receptor (IGF-1R) has surfaced as a significant target in multiple solid cancers due to its fundamental roles in pro-survival and anti-apoptotic signaling. However, development of resistance to IGF-1R blockade represents a significant hindrance and limits treatment efficacy in the clinic. In this study, we identified acquired resistance to IGF-1R blockade with R1507, an antibody against IGF-1R, and with BMS-754807, a small molecular inhibitor of IGF-1R/insulin receptor (IR). We showed that treatment with an IGF-IR antibody, R1507, or an IR/IGF-IR kinase inhibitor, BMS-754807, was associated with increased activation of YES/SRC family tyrosine kinase (SFK) in rhabdomyosarcoma (RMS). Combining anti–IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition–induced activation of YES/SFK and displayed advantageous antitumor activity in vitro and in vivo. Our data provide evidence that IGF-1R blockade results in activation of the YES/SRC family kinase bypass resistance pathway in vitro and in vivo. This may be of particular clinical relevance since both Yes and IGF components are overexpressed in RMS. Increased YES/SFK activation might serve as a clinical biomarker for predicting tumor resistance to IGF-1R inhibition. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for patients with RMS.

Dsh homolog DVL3 mediates resistance to IGFIR inhibition by regulating IGF-RAS signaling.

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.

Abstract IA33: Combination targeted therapy in pediatric sarcomas

Abstract Our group, and many others have documented a crucial role for IGFIR signaling in several pediatric sarcomas. These studies, along with the development of fully human IGFIR antibodies led to clinical testing of these blocking antibodies in rhabdomyosarcoma, Ewing's sarcoma, and osteosarcoma, along with a variety of other sarcomas. In general these single agent Phase 2 studies showed objective responses rates below 20%. Furthermore, even in responding patients, the duration of response is short-lived, typically less than 18 weeks. Thus the majority of patients do not have long term benefit from IGFIR blockade, indicating the presence of innate or acquired resistance to this therapeutic approach. We have been using xenografts of human rhabdomyosarcoma cell lines RD(embryonal) and RH30 (alveolar) to model both innate and acquired resistance to IGFIR blockade. Our xenograft models of rhabdomyosarcoma predicted short duration responses that mimicked the clinical observations, and we have been attempting to use these models to better understand how various signaling pathways in addition to IGFIR may interact and indeed be used as “by-pass” resistance pathways. Our hope is that this knowledge will lead to rational combination targeted therapy that we assume will be necessary for maximal clinical benefit. I will discuss one example of an additional signaling pathway that was activated upon IGFIR blockade, and how this information led to combination targeted therapy that holds therapeutic promise. We recently demonstrated that Src-Family Kinase (SFK) YES is highly expressed and functional in rhabdomyosarcomas. Since SFK signaling has been previously linked to IGF signaling in other tumor types, we evaluated the effect of IGFIR on YES activation. YES is rapidly activated upon IGFIR blockade using either a monoclonal antibody directed against the IGFIR or an IGFIR kinase inhibitor, suggesting that YES activation could play a role in resistance to IGFIR blockade. We next tested dual SFK and IGFIR inhibition and found that combined SFK and IGFIR inhibition led to more potent inhibition of both RD and RH30 rhabdomyosarcoma cell lines and perhaps most importantly led to increased apoptosis. Based on these promising in vitro effects, we evaluated the combination of IGFIR Ab, R1507 plus the SFK inhibitor, dasatinib, in our RD and RH30 xenografts. While both R1507 and dasatinib led to tumor growth inhibition as single agents, mice developed resistance to both single agents within 70-90 days of treatment. In contrast, the mice treated with the combination of R1507 and dasatinib did not develop resistance after 90 days and also showed enhanced tumor growth inhibition. This example highlights the need to better understand how signaling pathways interact so that one may predict consequences of therapeutic intervention using targeted agents and develop combinations that will undoubtedly be necessary to achieve the maximal clinical benefit of agents designed to inhibit specific signaling pathways. Citation Format: Lee J. Helman, Fernanda Arnaldez, Christine Heske, Xiaolin Wan, Choh Yeung. Combination targeted therapy in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA33.

A Phase II Pharmacodynamic Study of Preoperative Figitumumab in Patients with Localized Prostate Cancer

Activation of the insulin-like growth factor 1 receptor (IGF-IR) is implicated in prostate cancer development and progression. This study evaluated biologic and clinical effects of figitumumab, a fully human monoclonal IGF-IR antibody, in patients with localized prostate cancer. Eligible patients received figitumumab 20 mg/kg intravenously every 3 weeks for 3 cycles followed by prostatectomy. The primary endpoint was IGF-IR expression inhibition as assessed by immunohistochemistry. Sixteen patients were accrued. Median age was 63 years, median prostate-specific antigen (PSA) was 7.2 μg/L (range, 2.5-35), clinical stage was T1 in four patients and T2 in 12 patients, Gleason score ≤ 7 or >7 in 15 and one patients. Two patients received only 1 cycle (patient choice and grade III hyperglycemia). A PSA decline from baseline of ≥ 25% and ≥ 50% occurred in 15 (94%) and 5 (31%) of patients. Mean figitumumab concentration was 350.4 μg/mL (range, 26.3-492.8) in plasma and 51.3 μg/g (range, 27.4-79.6) in prostate tissue. Compared with pretreatment biopsies, IGF-IR expression decreased in the prostatectomy specimens in 14 of 16 patients. The mean IGF-IR immunohistochemistry visual score was 2.1 (SD = 0.6) in biopsy and 1.1 (SD = 0.5) in prostatectomy specimens (P < 0.0001). Androgen receptor expression was also decreased and there was a trend for a decrease in downstream IGF-IR signaling components. Figitumumab is biologically active in prostate cancer. PSA declines in treatment-naive patients were observed, potentially mediated by IGF-IR effects on androgen receptor expression. These results support the clinical relevance of IGF-IR signaling in prostate cancer and justify further clinical trials.

Highlights of This Issue

Several antagonistic IGF-1R monoclonal antibodies with differential IGF-blocking capability and receptor downregulation efficiency are in clinical development for solid tumors. Here Dong and colleagues report that combining two IGF-IR inhibitory antibodies with distinct epitopes and ligand-blocking mechanisms led to significantly improved ligand blockade and receptor downregulation, resulting in enhanced inhibition of IGF-IR signaling and tumor growth. Individual IGF-IR antibodies were also found to have limitations in their antitumor activity at high IGF levels, which can be overcome by the antibody combination. Their data suggest that antibodies targeting two distinct epitopes of IGF-IR may provide improved clinical efficacy.Development of drug resistance has been a growing concern for inhibitors of the class III receptor tyrosine kinase, FLT3, found in a subset of AML patients. Weisberg and colleagues identified the novel type II ATP competitive inhibitors, HG-7-85-01 and HG-7- 86-01, as highly potent and selective inhibitors of PKC412- sensitive and –resistant mutant FLT3 with the ability to synergize with PKC412 and standard chemotherapeutic agents. The ability of these compounds to inhibit mutant FLT3 is a distinctive feature that, in combination with demonstrated activity against a diverse panel of tyrosine kinase gatekeeper mutants, underscores their structural uniqueness and potential clinical versatility.Pancreatic cancer is a highly lethal malignancy driven by cancer stem cells (CSC). Rajeshkumar and colleagues identified a druggable target enriched in pancreatic CSCs (DR5). Gemcitabine, the first-line chemotherapeutic agent for pancreatic cancer, was ineffective in depleting CSCs and eventually culminated in tumor recurrence in pancreatic cancer xenografts. A combination of tigatuzumab, a fully humanized DR5 agonistic mAb, with gemcitabine resulted in remarkable reduction in pancreatic CSCs, tumor remissions, and long-term disease control in a model that is considered more difficult to treat. These findings have potential therapeutic implications for improving the current standard of care for pancreatic cancer patients.

Abstract 2329: Alterations in laminin expression modulate prostate cancer cell behavior through changes in integrin and insulin-like growth factor receptor actions

Abstract Introduction: During development of primary prostate carcinomas alterations to the extracellular matrix occur, including a loss of laminin 332 (LM332) and one of its cell surface receptors, β4 integrin. There are few published studies on what the laminin composition of metastases may be or how alterations in laminin expression affect prostate cancer cell behavior. Expression of the insulin-like growth factor receptor (IGF-IR) also is altered in prostate cancer progression and some studies suggest cross-talk between integrins and IGF-IR. Purpose: To determine if laminin and integrin expression is altered in prostate cancer metastases and how these alterations affect prostate cancer cell behavior in light of the interactions between integrins and IGF-IR. Methods: We performed immunofluorescent (IF) staining on tissue samples of primary PCa and metastases. We also used various prostate cancer cell lines, including M12 cells transfected with the LMα4, β2, or α4β2 chains, to perform adhesion, migration, and proliferation assays in the presence of inhibitory integrin and IGF-IR antibodies. Results: Our IF staining demonstrated that in addition to a loss in LM332 expression, the LMα4 and β2 chains were absent from the prostate basal membrane in primary prostate tumors. Expression of the LMα5 and β1γ1 chains was retained in primary tumors as well as in lymph node metastases. We saw a loss of β4 integrin in primary tumors as well as in metastatic cancer, but α6 and β1 integrins were retained; in lymph node mets, α2 and α3 integrins also were expressed. For M12 laminin cells, enhanced adhesion to both collagen and fibronectin was further increased by treatment with the inhibitory IGF-IR antibody A12. An inhibitory β1 integrin antibody, but not individual alpha integrin antibodies, blocked this enhancement. Inhibitory β1 as well as alpha integrin antibodies blocked adhesion of the empty-vector control cells. Similar results were obtained for cell migration studies using a scratch-assay. Staining of cells following exposure to A12 showed there was an increase in LM332 and LMβ1γ1 expression compared to untreated cells, which may account for the increased adhesion and migration following A12 treatment. We then examined laminin expression of various prostate cancer cell lines: LNCaP, LNCaP C4-2, PC3, and DU145. We found no expression of LM332 in any of these lines. Cytoplasmic expression of the LMβ1γ1 and LMβ2 (LNCaP lines) chains was detected, but deposition of these laminins was not present. These lines demonstrated increased adhesion to laminin-rich ECM compared with adhesion to fibronectin or collagen. And these lines showed enhanced proliferation when grown on laminin-rich ECM from the M12 laminin cells. Summary: Alterations in laminins affect the adhesion, migration, and proliferation of prostate cancer cells, in part, through integrin and IGF-IR actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2329.

Potential Benefit of an Agonistic Anti IGFIR Antibody (scFv-Fc) as an Effective Breast Cancer Therapy.

Abstract Multiple monoclonal antibodies targeting the type-1 insulin-like growth factor receptor (IGFIR) are currently in different phases of clinical trials. All the reported antibodies inhibit biochemical activation of IGFIR and block cell proliferation. In contrast, we have reported an agonistic anti-IGFIR antibody scFv-Fc that stimulates IGFIR and its downstream signaling of PI3K and MAPK pathways to promote cell proliferation in vitro. Like the purely antagonistic antibodies, our previous work has shown that scFv-Fc can down regulate IGFIR levels, partially inhibit xenograft growth of MCF-7 cells in athymic mice, and render MCF-7 cells refractory to the mitogenic effects of IGF-I after the initial wave of agonistic activity. MCF-7 cells maintained long term in scFv-Fc have low levels of IGFIR expression, yet the remaining receptor is activated and inhibited by an IGFIR tyrosine kinase inhibitor (TKI) AEW541. In this study, we have further explored the future potential of using this antibody as breast cancer therapy. The common characteristic effect of anti-IGFIR antibodies is to down regulate IGFIR. Since scFv-Fc can activate IGFIR signaling in a manner different than the natural ligand IGF-I, we examined if this antibody could render cells more sensitive to TKI. In MCF-7 cells, IC50 of the IGF1R tyrosine kinase inhibitor AEW541 was decreased 5 to 10 fold when cultured in scFv-Fc. ER negative MDA-MB-231 cells are insensitive to both inhibitory antibody (AVE1642) and TKI (AEW541) in the presence or absence of IGF-I, which may due to the very low levels of IGF1R that are weakly activated by IGF-I. MDA-MB-231 cells were not affected by AEW541 in monolayer growth assays. After chronic treatment of MDA-MB-231 with scFv-Fc for 2 months, AEW541 significantly inhibited monolayer cell growth by 30% in the presence or absence of IGF-I. We also examined the effect of scFv-Fc's effect on AEW541 inhibition of cell motility in MDA-MB-231 cells. Both acute and chronic treatment of scFv-Fc dramatically inhibited cell migration by 90%.Inhibitory antibodies AVE 1642 and MK 0646 had no effect on cell migration. Both IGF-I and excess amounts of AVE 1642/MK 0646 did not reverse scFv-Fc inhibition on cell migration, suggesting other cell signaling system may be involved. In MDA231-BO, a metastatic variant of MDA-MB-231, scFv-Fc had less inhibitory effect on basal cell migration, but in combination of AEW541 treatment, cell migration was significantly decreased by 70%. In conclusion, our data suggests an antibody with agonistic activity may have different properties than purely inhibitory antibodies. Specifically, initiating biochemical signaling followed by inhibition of tyrosine kinase activity and receptor downregulation may be a potent way to disrupt IGF mediated breast cancer cell biology. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3125.

C-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF–IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF–IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

MCF-7 cells selected for acquired resistance to an anti IGF-IR antibody remain sensitive to fulvestrant and to an IGFIR tyrosine kinase inhibitor (TKI).

Abstract Abstract #72 Monoclonal antibodies and tyrosine kinase inhibitors (TKI) targeting the type-1 Insulin-like growth factor receptor (IGFIR) are currently in clinical trials. Resistance to several targeted therapies has been observed and is not well understood. Estrogen receptor (ER) has been shown to interact with IGFIR signaling to promote cell growth. To examine acquired resistance to IGF1R monoclonal antibodies, we selected ER positive MCF7L cells in increasing concentrations of an IGFIR inhibitory antibody (scFvFc, treated over 2 years). These cells (MCF7L-scFvFc) had downregulated IGFIR levels, increased basal phosphorylation of IRS-1 with constitutive activation of Akt. MCF7L-scFvFc cells had enhanced basal growth and were no longer responsive to IGF-I and estradiol (E2). To examine the relationship between IGF1R and ER function, we used the pure steroidal anti-estrogen fulvestrant to examine effects on growth of the parent and antibody-resistance cells. In parent MCF7L cells, fulvestrant inhibited growth responses to IGF-I and E2 treatment when each ligand was given individually, but when both ligands were given together, the growth inhibition was reversed. In MCF7L-scFvFc, fulvestrant inhibited growth response to IGF-I, but cells still responded to E2 even in the presence of fulvestrant. Fulvestrant inhibition of MCF7L-scFvFc basal growth was associated with downregulation of ER and IGFIR system components. Fulvestrant treatment induced PARP cleavage in MCF7L, but not in MCF7L-scFvFc cells. IGF-I partially protected cells against fulvestrant induced PARP cleavage, and E2 treatment has full protective effects on these cells. These data suggest that MCF7L-scFvFc cells were more resistant to cell apoptosis induced by fulvestrant. Other monoclonal antibodies directed against IGF1R were ineffective in reversing the resistance in MCF7L-scFvFc cells. To determine if activated IGF1R was responsible for the behavior of MCF7L-scFvFc cells, we used the IGF1R TKI AEW541. AEW541 inhibited growth of these antibody resistant cells and a combination of fulvestrant and AEW541 completely inhibited cell growth and IGF signaling in both MCF7L and MCF7L-scFvFc cells. Thus, activated IGFIR signaling was still required for the growth of MCF7L-scFvFc cells. In conclusion, MCF7L-scFvFc had low levels of IGF1R but retained activated IGF1R signaling that was inhibited by TKI (AEW541). In addition, the ER antagonist fulvestrant also inhibited growth of these cells. In these ER-positive cells, combined blockade of ER and IGF1R could provide a more prolonged inhibition of cell growth. Moreover, cells selected for resistance to an IGF1R antibody remain sensitive to IGF1R TKIs. Thus, concurrent or sequential targeting of IGF1R and ER function should be examined in breast cancer clinical trials of ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 72.

The insulin-like growth factor receptor I status in luminal A breast carcinoma.

Abstract Abstract #4034 Background: The IGF-IR overexpression is reported to be detectable in 43-50% of primary breast cancers, its clinical and prognostic significance remains undetermined. Insulin growth factor type I (IGF-IR) is induced by estrogens in estrogens receptor (ER) positive cells that could explain why the IGF-IR overexpression is strongly related to ER positive breast cancer. However a small proportion of ER/PR negative cases overexpressed IGF-IR. The aim of this study was to correlate IGF-IR status by three techniques on a series of breast carcinomas : DNA microarrays ,CGH arrays and Immunohistochemistry.&amp;#x2028; Material and Methods: Immunohistochemical phenotypes were studied in a series of 72 cases of breast carcinoma (40 basal and 32 luminal A). A series of 13 Markers was also studied IGF-IR, ER, PR, AR441, bcl2, FoxA1, Ki67, Gata3, P53, EGFR, ERBB2, CAV1 and MS110(BRCA1). Gene expression profiling with DNA microarrays (Affymetrix) and 244k CGH microarray was done on the same cases.&amp;#x2028; Results: The IGF-IR expression was observed in IHC in 61% of the cases, overexpression in 25% and Gain or amplification in 7% .The IHC IGF-IR positivity was strongly correlated to the IGF-IR expression ( R=0.28 p&amp;lt;0.016) and IGF-IR DNA status (R=0.53 p= 5.610-6 ). IGF-IR over-expression in DNA microarrays is the only one results to be strongly associated to the luminal A subtype : 15/18 of the over expressed cases p=1.56 e-7. However the IHC expression of the ER related biomarkers is significantly associated to the IGF-IR overexpression.&amp;#x2028; &amp;#x2028; Discussion: The IGF-IR overexpression is representative of the Luminal A subtype, and to the ER regulated gene in IHC. Exploration of a panel of IGF-IR antibody is mandated before to best evaluate this target gene in IHC, who could contribute to the hormonal response. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4034.

Addiction to Elevated Insulin-like Growth Factor I Receptor and Initial Modulation of the AKT Pathway Define the Responsiveness of Rhabdomyosarcoma to the Targeting Antibody

Insulin-like growth factor I receptor (IGF-IR) and its ligands are overexpressed by tumors, mediating proliferation and protecting against stress-induced apoptosis. Accordingly, there has been a considerable amount of interest in developing therapeutic agents against IGF-IR. IGF-IR is believed to be ubiquitously expressed without detectable mutation or amplification in cancer. We explored the determinants of cellular response to a humanized anti-IGF-IR antibody. Our results showed a large variation in IGF-IR levels in rhabdomyosarcoma tumor specimens that were comparable with those in rhabdomyosarcoma cell lines. In vitro analysis revealed a direct and very significant correlation between elevated IGF-IR levels and antiproliferative effects of the antibody and defined a receptor number that would predict sensitivity. Our data further suggested a strong dependence on IGF-IR for AKT signaling in cells with elevated IGF-IR. The sensitivity of the high IGF-IR-expressing cells was blocked with a constitutively active AKT. The extracellular signal-regulated kinase pathway was not affected by the antibody. In vivo studies showed that anti-IGF-IR had single-agent antitumor activity; furthermore, predictions of responses based on IGF-IR levels were accurate. In vivo biomarker analysis suggested that h7C10 down-regulated both IGF-IR and p-AKT initially, concordant with antitumor activity. Subsequent progression of tumors was associated with reactivation of p-AKT despite sustained suppression of IGF-IR. These results identified the first predictive biomarker for anti-IGF-IR therapies in cancer.

Cardiomyoblast apoptosis induced by insulin-like growth factor (IGF)-I resistance is IGF-II dependent and synergistically enhanced by angiotensin II

This study explores the synergistic effect of cardiomyoblast apoptosis induced by angiotensin II (Ang II) and Insulin-like growth factor (IGF)-I resistance, and elucidates the role of IGF-II via IGF-II receptor (R) and calcineurin pathways in apoptosis induced by Ang II and IGF-I resistance. Apoptosis of cultured cardiomyoblast H9c2 cells was assessed by DNA fragmentation on agarose gel electrophoresis, nuclear condensation stained with DAPI, and Western blot analysis of pro-apoptotic Bad and cytochrome c in various combinations of control, Ang II, antisense IGF (I or II), IGF (I or II) antibody, IGF (I or II) receptor (R) antibody, or calcineurin inhibitor (Cyclosporine A, (CsA)). We found the following: (1) The combination of Ang II and IGF-I deficiencies had a synergistic effect on apoptosis, confirmed by DNA fragmentation, nuclei condensation, and increases in such pro-apoptotic proteins as Bad, cytochrome c, caspase 9, and caspase 3 in H9c2 cells. (2) IGF-II and IGF-IIR protein products were increased by antisense IGF-I and IGF-I resistance, but these IGF-II protein products were not affected by sense IGF-I and non-specific antibody IgG in H9c2 cells. (3) The alteration of Bad protein level and the release of cytochrome c, both induced by treatments containing combinations of Ang II and antisense IGF-I, IGF-I antibody or IGF-IR antibody, were inhibited by IGF-II antibody. (4) DNA fragmentation, Bad, and cytochrome c which was induced by treatments combining IGF-IR antibody with Ang II or combining IGF-IR antibody with IGF-II were remarkably attenuated by CsA. IGF-I deficiency and/or IGF-IR resistance induced apoptosis in cardiomyoblast cells. The apoptosis, which might have been caused by the upregulation of IGF-II and IGF-IIR genes possibly activated the downstream calcineurin pathway, was synergistically augmented by Ang II.

Influence of IGF-IR stimulation or blockade on proliferation of human renal cell carcinoma cell lines

Several tumors secrete insulin-like growth factors (IGFs) for autocrine growth stimulation and protection from apoptosis. However, the mechanisms responsible for tumor growth in renal cell carcinoma (RCC) are unclear. In this study the biological role of exogenous IGFs in two malignant RCC cells (Caki-2 and SK-RC-52) were investigated in vitro, and compared to the breast cancer cell line MCF-7. IGFs but not the related epidermal growth factor stimulated both RCC cell lines. Caki-2 expressed higher levels of IGF-IR and proliferated more vigorously to added IGF-I, IGF-I analogues des(1-3)IGF-I, LongR3IGF-I and IGF-II compared to SK-RC-52. Neutralizing IGF-IR antibodies reduced the IGF driven proliferation in both cell lines. Interestingly, soluble IGF-I receptor resulted in strong growth inhibition of SK-RC-52, while no marked effect on Caki-2 was observed. Moreover, Caki-2 expressed a broad panel of IGFBPs, while SK-RC-52 more selectively secreted high levels of IGFBP-3. Exogenous IGFBP-3 strongly inhibited IGF-I driven proliferation in SK-RC-52, but worked in synergy with IGF-I in Caki-2. Both the IGF-IR and IGFBP-3 were present in respectively 4/4 and 4/8 human malignant renal tissues. In light of this and with the functional data presented in this study, interference with this growth factor system may provide a novel therapeutic approach in renal cancer therapy.

Expression of IGR-IR and VEGF and Trophoblastic Proliferative Activity in Placentas from Pregnancies Complicated by IUGR

Intrauterine growth retardation (IUGR) is recognized as an important cause of low birth weight and elective preterm delivery. IUGR is associated with multiple causative factors, including placental dysfunction. The aim of this prospective study was to investigate the role of trophoblastic proliferative activity and type I insuline-like growth factor receptor (IGF-IR) and vascular endothelial growth factor (VEGF) expressions in the pathogenesis of IUGR. Immunohistochemistry using VEGF, IGF-IR, and Ki-67 antibodies was performed on formalin-fixed placental tissues of third-trimester pregnancies complicated by IUGR (n = 19) and pregnancies with appropriately grown fetuses (n = 27). In addition, histopathological examination of the placentas was performed, and histological findings were categorized into three groups: utero-placental vascular pathologies (UPVP), coagulation-related pathologies, and chronic inflammation. Statistical analysis revealed that villous trophoblastic IGF-IR immunostaining was significantly weaker in placentas with IUGR (p < 0.001), whereas trophoblastic Ki-67 proliferative index and VEGF immunoscoring did not show any significant difference. Histologically, UPVP and chronic inflammation were significant findings in placentas with IUGR (p = 0.04 and p = 0.04, respectively). In addition, placentas were significantly smaller in the IUGR group (p < 0.001). We conclude that villous trophoblastic IGF-IR expression may play a significant role in the pathogenesis of IUGR, and histopathological examination of placentas in pregnancies complicated by IUGR may yield significant findings. In contrast, based on our findings, trophoblastic proliferation and VEGF expression are unlikely to be significant parameters in the pathogenesis of IUGR.