IGF Ligands Research Articles

7942 Evolution of Insulin, Insulin-like Growth Factor and Their Cognate Receptors in Vertebrates, Invertebrates and Viruses

Abstract Disclosure: M. Chrudinova: None. Y.D. Dogru: None. R. Huang: None. R. Reiners: None. P. De Meyts: None. J. DaCosta: None. E. Altindis: None. The insulin/insulin-like growth factor (IGF) signaling system is an evolutionarily conserved system and controls central processes including metabolism, cellular growth, proliferation, and differentiation in vertebrates. On the other hand, invertebrate insulin-like peptides (ILPs) regulate growth, stress responses, longevity and sex-related functions. In addition, we recently showed that six viruses possess viral insulin-like peptides (VILPs) that can bind to the receptors and stimulate post-receptor signaling. The evolution of these hormones and the origin of VILPs are not completely understood. Here, we collected vertebrate and invertebrate insulin, IGF and their cognate receptor sequences to analyze their evolutionary relations. We analyzed 177 vertebrate insulins, 119 vertebrate IGF-1s, 127 vertebrate IGF-2s, 197 invertebrate ILPs and 6 viral insulin/IGF-1 like peptides. We also analyzed 214 insulin receptor (IR), 160 IGF receptor (IGF1R) and 39 invertebrate receptor sequences to identify conservation and the evolutionary trajectory to reveal conserved motifs. Through phylogenetic analyses, we observed a separate branching of invertebrate ILPs and vertebrate insulins, IGF-1s and IGF-2s with notable exceptions including VILPs. IGF-1 and IGF-2 ligands were evolutionally better conserved compared to insulin ligands. Insulin C-peptide which is processed and cleaved was not well conserved compared to the functional A- and B-chain residues. In contrast, C-peptide of IGF-1 that plays an important role in receptor binding was very well conserved. IGF-1 C-domain showed higher variability in IGF1R binding residues compared to the A and B-domains, while residues important for binding of IGF-2 to IGF1R were well conserved. Insulin residues interacting with IR binding Site 1 were more conserved than those interacting with IR binding Site 2, while residues important for insulin dimerization and hexamerization residues are well conserved across species. We also showed that VILPs contain 14 amino acids that are conserved in insulin and IGFs, indicating their functional importance. Phylogenetic relationships of the receptor sequences suggest vertebrate IRs and IGF1Rs evolved from a common ancestor during early vertebrate evolution. Interestingly, receptor subunits revealed a closer relation of amphibian IR α-subunits to Mammalia IR α-subunits than Aves and Reptilians. The conservation rate of receptor residues essential for ligand-receptor interaction revealed that these residues are best conserved in the L1 domain for IR and IGF1R, with a higher rate of variation in other domains for both receptors. Taken together, our study provides a comprehensive analysis of insulin, IGFs, VILPs and their cognate receptors’ evolution, and opens new avenues to better understand the evolution, function, and structure relations of this complex signaling system. Presentation: 6/3/2024

Targeting IGF-IR improves neoadjuvant chemotherapy efficacy in breast cancers with low IGFBP7 expression

There has been a long-standing interest in targeting the type 1 insulin-like growth factor receptor (IGF-1R) signaling system in breast cancer due to its key role in neoplastic proliferation and survival. However, no IGF-1R targeting agent has shown substantial clinical benefit in controlled phase 3 trials, and no biomarker has been shown to have clinical utility in the prediction of benefit from an IGF-1R targeting agent. IGFBP7 is an atypical insulin-like growth factor binding protein as it has a higher affinity for the IGF-1R than IGF ligands. We report that low IGFBP7 gene expression identifies a subset of breast cancers for which the addition of ganitumab, an anti-IGF-1R monoclonal antibody, to neoadjuvant chemotherapy, substantially improved the pathological complete response rate compared to neoadjuvant chemotherapy alone. The pCR rate in the chemotherapy plus ganitumab arm was 46.9% in patients in the lowest quartile of IGFBP7 expression, in contrast to only 5.6% in the highest quartile. Furthermore, high IGFBP7 expression predicted increased distant metastasis risk. If our findings are confirmed, decisions to halt the development of IGF-1R targeting drugs, which were based on disappointing results of prior trials that did not use predictive biomarkers, should be reviewed.

Abstract PO3-24-09: May ESR1 mutant breast cancer cells influence fibroblast phenotype?

Abstract For almost 50 years, it has been postulated that carcinogenesis relies on the intrinsic genetic abnormalities of tumor cells, but in the last decades, it has been reported that their functional interactions with the tumor microenvironment (TME) might affect therapy response and contribute to the disease progression. Based on these observations, the aim of the present study was to investigate the functional interactions between one of the essential components of TME, the fibroblasts, and breast cancer (BC) cells, expressing mutations in the hormone-binding domain (HBD) of the gene encoding for the estrogen receptor alpha (ESR1). Thus, we employed estrogen receptor-positive MCF-7 BC cells CRISPR engineered to express the Y537S-HBD mutation. Y537S is the most commonly detected mutation harbored in HBD-ESR1 and is a major contributor to endocrine therapy resistance and poor clinical outcome in BC. We collected conditioned media (CM) from BC cells and fibroblasts isolated from primary human tissues (normal fibroblasts, NFs, and cancer-associated fibroblasts, CAFs) in order to assess in vitro co-culture systems that can mimic the complex in vivo TME. It was interesting to observe that CAFs displayed a significant increase in their proliferation and migration when exposed to CM of mutant BC cells with respect to the parental ones. These latter findings fit with the immunofluorescence analysis showing a more evident formation of actin stress fibers. However, proteomic analysis demonstrated, in the same circumstances, more significant changes in NFs than in CAFs. Among the up-regulated proteins revealed by NFs upon Y537S-CM exposure, most of them overlap the up-regulated proteins featuring the intrinsic phenotype of CAFs, mainly involved in the cellular ultrastructural organization (i.e. cytoskeleton, focal adhesion, vesicle, and organelle) as evidenced by Metacore functional analysis. On the other side, we demonstrated that mutant clones showed more aggressive behavior in terms of proliferation, growth, migration, and invasion, compared to the parental counterpart upon exposure to the CM-derived from NFs and CAFs. This led us to further investigate the intrinsic properties of the mutated clones through their genomic and proteomic profiles that evidenced, by using Metacore software, an enrichment of insulin-like growth factor 1 receptor (IGF1R) pathway. The relevance of the latter datum was confirmed by the higher content of the IGF-1 ligand found in the secretome of mutant cells. Thus, the short autocrine loop maintaining IGF1/IGF1R signaling activation in mutant clones might reasonably play an important role in mediating the autocrine and paracrine effects between mutant cells and fibroblasts. For instance, the pharmacological blockade of IGF1R signaling interfered with the reciprocal interactions between fibroblasts and BC cells that sustained tumor growth and progression as evidenced in both “in vitro” and “in vivo” experiments. In conclusion, our study addressed the IGF1R pathway as a potential druggable target helpful to disconnect mutant breast cancer cell-fibroblast crosstalk that potentiates breast tumor growth and progression. Citation Format: Luca Gelsomino, Amanda Caruso, Rocco Malivindi, Adel Elisabetta Leonetti, Emine Tasan, Salvatore Panza, Giuseppina Daniela Naimo, Ines Barone, Cinzia Giordano, Daniela Bonofiglio, Loredana Mauro, Guowei Gu, Suzanne Fuqua, Stefania Catalano, Sebastiano Andò. May ESR1 mutant breast cancer cells influence fibroblast phenotype? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-09.

Abstract 7351: Delineating the prostate cancer tumor microenvironment through scRNA-seq profiling at different timepoints

Abstract Distant metastasis remains the leading cause of death in patients with prostate cancer (PCa), however, the underlying mechanisms of this process remain unclear. Using samples from genetically-engineered mouse models (GEMMs) of PCa, we have identified eight different stromal cell states linked to epithelial mutations that may impact PCa progression to metastatic stages. To further investigate the unique intercellular interactions in the tumor microenvironment (TME) involved in mediating disease progression, we use single cell RNA sequencing (scRNA-seq) to profile primary PCa samples from different GEMMs including TRAMP, Hi-MYC, and FVBN, at different timepoints. Analysis of the scRNA-seq data revealed a significant enrichment of three clusters, designated as stromal c6 (s6), epithelial c7 (e7), and epithelial c10 (e10), exclusively in TRAMP, each expressing proliferative markers such as Mki67 and Top2a. To investigate the intercellular interactions among these clusters, we conducted ligand-receptor (LR) interaction analysis using CellChat. Notably, s6 is expressing the genes encoding ligands Bmp4, Fn1, and Igf1, which interact with the receptors Bmpr1b/Bmpr2, Itga3/Itgb1, and Igf1r, respectively expressed in e7. Subsequently, the transcriptomic profiles of these cells were used to build and validate a robust gene signature that can predict metastasis and survival in patients with PCa using a large cohort of 1239 samples, 930 of which were used for training while the remaining 309 samples were used for testing the model’s performance. In conclusion, these results underscore the role of the TME mediating PCa progression and highlight the potential of leveraging the TME to extract predictive and prognostic features which can be later employed to build predictive and prognostic models for patients with PCa. Citation Format: Itzel Valencia, Hubert Pakula, Mohamed Omar, Massimo Loda, Luigi Marchionni. Delineating the prostate cancer tumor microenvironment through scRNA-seq profiling at different timepoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7351.

Glycan Modulation of Insulin-like Growth Factor-1 Receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.

Glycan Modulation of Insulin‐like Growth Factor‐1 Receptor

AbstractThe insulin‐like growth factor‐1 receptor (IGF‐1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF‐1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG‐protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF‐1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF‐1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti‐cancer drug discoverers.

Biological effects and regulation of IGFBP5 in breast cancer.

The insulin-like growth factor receptor (IGF1R) pathway plays an important role in cancer progression. In breast cancer, the IGF1R pathway is linked to estrogen-dependent signaling. Regulation of IGF1R activity is complex and involves the actions of its ligands IGF1 and IGF2 and those of IGF-binding proteins (IGFBPs). Six IGFBPs are known that share the ability to form complexes with the IGFs, by which they control the bioavailability of these ligands. Besides, each of the IGFBPs have specific features. In this review, the focus lies on the biological effects and regulation of IGFBP5 in breast cancer. In breast cancer, estrogen is a critical regulator of IGFBP5 transcription. It exerts its effect through an intergenic enhancer loop that is part of the chromosomal breast cancer susceptibility region 2q35. The biological effects of IGFBP5 depend upon the cellular context. By inhibiting or promoting IGF1R signaling, IGFBP5 can either act as a tumor suppressor or promoter. Additionally, IGFBP5 possesses IGF-independent activities, which contribute to the complexity by which IGFBP5 interferes with cancer cell behavior.

Abstract 2653: Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor

Abstract Glycosaminoglycans (GAGs), such as heparan sulfate (HS), have been implicated in tumor initiation, progression and metastasis. Earlier we have shown that a defined GAG sequence of six residues, but not four or eight residues, inhibits colorectal cancer stem cells (CSCs) by inducing activation of p38 MAPK. To transform this sequence into a drug-like molecule, we developed a synthetic mimetic of the GAG sequence, labeled as G2.2, which was also found to selectively target CSCs over bulk adherent tumor cells. Unfortunately, G2.2’s oral bioavailability was low. To improve upon its drug-like properties, we pursued a hypothesis-driven analog design to derive three lipid-modified analogs (LMAs). Across a panel of > 15 patient-derived colorectal cancer (CRC) cell lines, which could be stratified according to their clinically-relevant consensus molecular subtypes, LMAs showed enhanced potency while retaining selectivity against CSCs in spheroid inhibition assays. Both G2.2 and LMAs displayed better inhibition of cell lines with mesenchymal phenotypes and metabolic dysregulation. Microarray-based screening against more than a dozen receptor tyrosine kinases led to identification of IGF1R as a potential receptor of the synthetic GAG mimetics. To ascertain these screening results, biophysical studies were performed to clarify preferred soluble and/or cell surface target receptors. In line with microarray results, G2.2 preferentially bound to IGF1R in comparison to its soluble ligand IGF-1. G2.2 also preferred IGF1R as compared to an alternative receptor FGFR1. Further, LMAs bound to IGF1R with improved affinities as compared to parent mimetic G2.2, which could explain anti-CSC inhibition results. Unexpectedly, detailed fluorescence titrations revealed that IGF1R affinity of LMAs is minimally impacted by salt concentration, suggesting that these GAG mimetics utilize non-ionic forces in binding, which support the selectivity of target engagement. Overall, this work proves a powerful proof of concept that synthetic GAG mimetics, such as G2.2 and LMAs, present a unique class of anti-cancer therapeutics with high potential for selective elimination of the tumor initiating subpopulation of CRC cells in patient tumors. Citation Format: Connor O'Hara, Shravan Morla, Ravikumar Ongolu, Nirmita Patel, Rio Boothello, Bhaumik Patel, Umesh Desai. Synthetic, small molecule glycosaminoglycan mimetics induce novel anti-cancer activity through preferential targeting of a growth factor receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2653.

Ganitumab and metformin plus standard neoadjuvant therapy in stage 2/3 breast cancer

I-SPY2 is an adaptively randomized phase 2 clinical trial evaluating novel agents in combination with standard-of-care paclitaxel followed by doxorubicin and cyclophosphamide in the neoadjuvant treatment of breast cancer. Ganitumab is a monoclonal antibody designed to bind and inhibit function of the type I insulin-like growth factor receptor (IGF-1R). Ganitumab was tested in combination with metformin and paclitaxel (PGM) followed by AC compared to standard-of-care alone. While pathologic complete response (pCR) rates were numerically higher in the PGM treatment arm for hormone receptor-negative, HER2-negative breast cancer (32% versus 21%), this small increase did not meet I-SPY’s prespecified threshold for graduation. PGM was associated with increased hyperglycemia and elevated hemoglobin A1c (HbA1c), despite the use of metformin in combination with ganitumab. We evaluated several putative predictive biomarkers of ganitumab response (e.g., IGF-1 ligand score, IGF-1R signature, IGFBP5 expression, baseline HbA1c). None were specific predictors of response to PGM, although several signatures were associated with pCR in both arms. Any further development of anti-IGF-1R therapy will require better control of anti-IGF-1R drug-induced hyperglycemia and the development of more predictive biomarkers.

Pregnancy-associated plasma protein A mRNA expression as a marker for differentiated thyroid cancer: results from a \u201csurgical\u201d and a \u201ccytological\u201d series

PurposePregnancy-associated plasma protein A (PAPPA) is a metalloproteinase initially described for its role during pregnancy. PAPPA regulates IGF ligands 1 (IGF1) bioavailability through the degradation of IGF-binding protein 4 (IGFBP4). After the cleavage of IGFBP4, free IGF1 is able to bind IGF1 receptors (IGF1R) triggering the downstream signaling. Recently, PAPPA expression has been linked with development of several cancers. No data have been published on thyroid cancer, yet.MethodsWe evaluated PAPPA, insulin-like growth factor (IGF1), IGF1 receptors (IGF1R) and IGF-binding protein 4 (IGFBP4) mRNA expression levels in a “Surgical series” of 94 thyroid nodules (64 cancers, 16 follicular adenomas and 14 hyperplastic nodules) and in a “Cytological series” of 80 nodules from 74 patients underwent to fine-needle aspiration cytology (FNAC). In tissues, PAPPA was also evaluated by western blot.ResultsWe found that PAPPA expression was increased in thyroid cancer specimen at mRNA and protein levels and that, adenomas and hyperplastic nodules had an expression similar to normal tissues. When applied on thyroid cytologies, PAPPA expression was able to discriminate benign from malignant nodules contributing to pre-surgical classification of the nodules. We calculated a cut-off with a good specificity (91%) which reached 100% when combined with molecular biology.ConclusionThese results show that PAPPA could represent a promising diagnostic marker for differentiated thyroid cancer.

Altered redox homeostasis in steroid-depleted follicles attenuates hCG regulation of follicular events: Cross-talk between endocrine and IGF axis in maturing oocytes

Steroids and insulin-like growth factors (Igfs) are indispensable for folliculogenesis and reproductive fitness in the vertebrate ovary. The intrafollicular redox balance is also of immense importance for ovarian follicles wherein low levels of ROS are being utilized for cell signalling and regulation of gene expression; its excess may interfere with normal physiological processes leading to ovotoxicity. However, the functional relevance of ovarian steroidogenesis in maintaining the follicular microenvironment with coordinated redox homeostasis and intra-ovarian growth factors axis is relatively less understood. Using zebrafish full-grown (FG) ovarian follicles in vitro, our study shows that blocking steroid biosynthesis with anti-steroidal drugs, DL-aminoglutethimide (AG) or Trilostane (Trilo), prevents hCG (LH analogue)-induced StAR expression concomitant with a robust increase in intrafollicular ROS levels. Congruent with heightened intracellular levels of superoxide anions (O2•–) and hydrogen peroxide (H2O2), priming with AG or Trilo abrogates the transcript abundance of major antioxidant enzyme genes (SOD1, SOD2, and CAT) in hCG-stimulated follicles. Significantly, blocking steroidogenesis attenuates transcript abundance of HSP70 but elevates NOX4 expression potentially through ERα-mediated pathway. Importantly, disrupted redox balance in AG/Trilo pre-incubated FG follicles negatively impacts hCG-mediated activation of PKA/CREB signaling and transcriptional activation of igf ligands. Elevated ROS attenuation of antioxidant defense parameters and impaired endocrine and autocrine/paracrine homeostasis converge upon reduced p34cdc2 (Thr-161) phosphorylation, a reliable marker for MPF activation, and resumption of meiotic G2-M1 transition in hCG-treated follicles. Collectively, altered redox homeostasis in steroid-depleted follicles has a significant negative influence on GTH (LH) regulation of follicular events, specifically Igf synthesis, meiotic maturational competence and ovarian fitness.

Abstract 2357: Anticancer effects of calorie restriction in a murine C3-TAg model of triple-negative breast cancer: the role of miR-15b

Abstract Calorie restriction (CR) has an antitumorigenic effect against triple-negative breast cancer (TNBC). A key mitogenic pathway modulated by CR involves insulin-like growth factor (IGF)-1, which plays a central role in local and systemic growth and cell survival by activating the PI3K/AKT/mTOR pathway downstream of the IGF-1 receptor (IGF-1R). Although multiple miRNAs that target components of the IGF-1 signaling pathway have been identified, the impact of CR on IGF-1-related miRNA expression has not been addressed. We hypothesize that CR-induced decreases in TNBC development and progression involve miRNA's regulating IGF-1 signaling, we used C3(1)/SV40 T-antigen (C3-TAg) mice (a TNBC model), a C3-Tag progression series of cell lines (M28, M6, and M6C), and human TNBC cell line (MDA-MB-231). The expression of miRNAs previously reported to target components of the IGF-1 and/or mTOR pathways was evaluated by real-time PCR. Hormone levels were measured using the mouse Luminex Screening Assay. To mimic CR in vitro, and to test the effects of miRNA manipulation, the C3-Tag series of TNBC cell lines were treated (separately and in combination) with serum restriction, BMS754807 (inhibitor of IGF1-R), and various levels of recombinant IGF-1 as well as manipulated levels of miRNA's associated with IGF-1/mTOR signaling. We also conducted in silico analysis using Metabric, TCGA, TargetScan, and miRTarBase data sets to identify genes and pathways associated with specific miRNAs expression related to the PI3K/Akt/mTOR pathway. Our results confirm that CR maintains lower body weight, reduces circulating levels of IGF-1, and slows spontaneous TNBC development and progression in mice. CR also significantly increased the expression of miR-15b and miR-486. In vitro studies showed miR-15b and miR-486 expression decreased with increasing tumorigenicity of the C3-tag series of TNBC progression. Treatment of mouse and human TNBC cells with low IGF-1 induced miR-15b expression and inhibited proliferation. Moreover, miR-15b overexpression inhibited cell proliferation and decreased IGF-1R expression (mRNA and protein). Consistent with these results, the in silico analysis shows that the amplification of miR-15b correlates with downregulation of pathways involved in tumor growth and IGF1-R signaling. Together these findings suggest that reduced circulating IGF-1 levels in response to CR leads to the upregulation of miR-15b, which correspondingly targets and downregulates IGF-1R, both at the mRNA and protein levels. This combination of reduced IGF-1 ligand and miR-15b-induced IGF-1R downregulation contributes to the potent anticancer effects of CR and reveals potential targets for pharmacologically mimicking those effects. This research is supported by R35CA197627. Citation Format: Ximena Minerva Bustamante-Marin, Kaylyn L. Devlin, Om Dave, Jenna L. Merlino, Shannon B. McDonell, Michael F. Coleman, Stephen D. Hursting. Anticancer effects of calorie restriction in a murine C3-TAg model of triple-negative breast cancer: the role of miR-15b [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2357.

Growth Hormone/Insulin Growth Factor Axis in Sex Steroid Associated Disorders and Related Cancers.

To date, almost all solid malignancies have implicated insulin-like growth factor (IGF) signalling as a driver of tumour growth. However, the remarkable level of crosstalk between sex hormones, the IGF-1 receptor (IGF-1R) and its ligands IGF-1 and 2 in endocrine driven cancers is incompletely understood. Similar to the sex steroids, IGF signalling is essential in normal development as well as growth and tissue homoeostasis, and undergoes a steady decline with advancing age and increasing visceral adiposity. Interestingly, IGF-1 has been found to play a compensatory role for both estrogen receptor (ER) and androgen receptor (AR) by augmenting hormonal responses in the absence of, or where low levels of ligand are present. Furthermore, experimental, and epidemiological evidence supports a role for dysregulated IGF signalling in breast and prostate cancers. Insulin-like growth factor binding protein (IGFBP) molecules can regulate the bioavailability of IGF-1 and are frequently expressed in these hormonally regulated tissues. The link between age-related disease and the role of IGF-1 in the process of ageing and longevity has gained much attention over the last few decades, spurring the development of numerous IGF targeted therapies that have, to date, failed to deliver on their therapeutic potential. This review will provide an overview of the sexually dimorphic nature of IGF signalling in humans and how this is impacted by the reduction in sex steroids in mid-life. It will also explore the latest links with metabolic syndromes, hormonal imbalances associated with ageing and targeting of IGF signalling in endocrine-related tumour growth with an emphasis on post-menopausal breast cancer and the impact of the steroidal milieu.

Abstract PD7-08: Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models

Abstract Background: Deciphering the molecular landscape of resistance to the CDK4/6 inhibitors represents a critically important question for patients with hormone-receptor positive (HR+) metastatic breast cancer (MBC). Emerging insights from sequencing efforts suggest that inactivating alterations in the RB1 tumor suppressor occur in a small minority of patients and that a variety of heterogeneous mediators provoke resistance in patient samples. Proteins implicated in CDK4/6i resistance include cell cycle regulators such as cyclin E1/2, CDK6, and aurora kinase as well as known oncogenic signal transduction mediators involved in activation of the RAS-MEK and AKT-mTOR pathways. The insulin-like growth factor 1 receptor (IGF1R) has been implicated in modulating anti-estrogen resistance, and IGF1R inhibitors are currently in various stages of pre-clinical and clinical development. Methods: We identified patients with amplification events in IGF1R from a database containing targeted sequencing of solid tumor samples obtained from patients with HR+ MBC enrolled on a research biopsy protocol. Tumor biopsies may have been obtained at various points during each patient’s clinical treatment course. HR+ T47D cells were modified to over-express IGF1R via lentiviral infection and selection. Derivative cell lines were treated with IGF-1 ligand and downstream activation of the PI3K/AKT and RAS/MEK pathways were assessed via western blotting. Control cells (expressing GFP) were mixed with IGF1R-expressing cells 1:1 and cultured in the presence of IGF-1 ligand and palbociclib or other drugs, for 1-3 weeks. At the timepoint of interest, cells were harvested and the relative proportion of GFP or IGF1R-expressing cells were interrogated via flow cytometry. Results: We identified seven patients with HR+ MBC and IGF1R amplifications via targeted sequencing of tumor biopsies. Five of these patients had exposure to CDK4/6i-based therapy in the metastatic setting. Three patients demonstrated intrinsic resistance to CDK4/6i treatment (with duration <6 months) and biopsies were obtained prior to CDK4/6i exposure or, in one case, while on treatment. In an additional patient, after nine months of CDK4/6i-based therapy, an IGF1R amplification was present at the time of progression. In one counter-example, a baseline biopsy revealed IGF1R amplification and subsequent clinical benefit with CDK4/6i, exceeding 10 months, was noted. T47D cells over-expressing IGF1R demonstrated increased pERK and pAKT activation following introduction of IGF-1 ligand. Control GFP and IGF1R-expressing cells were plated 1:1 and cultured in the presence of IGF-1 ligand and palbociclib. In a flow cytometry-based competition assay, an IGF-1 dose-dependent increase in the relative proportion of IGF1R-expressing cells was noted after one, two, and three weeks of palbociclib treatment. The extent of IGF1R-expressing cell enrichment was attenuated in the presence of either a MEK inhibitor or an IGF1R inhibitor. Conclusions: IGF1R amplification events were identified in tumor biopsy samples that reflect either intrinsic or acquired resistance to CDK4/6i-based therapy. HR+ breast cancer cells which over-express IGF1R demonstrate enrichment under palbociclib drug selection in a flow cytometry-based competition assay, which was abrogated by concurrent use of a MEK or IGF1R inhibitor. These results suggest that IGF1R may join the increasingly heterogeneous landscape of CDK4/6i resistance mediators. Further exploration of this possibility is warranted. A subset of patients with IGF1R-mediated CDK4/6i resistance could benefit from therapeutic strategies designed to downregulate MEK or IGF1R activity. Citation Format: Seth A. Wander, Pingping Mao, Maxwell R. Lloyd, Gabriela N. Johnson, Kailey Kowalski, Utthara Nayar, Lillian M. Guenther, Kimberly Stegmaier, Eric P. Winer, Nancy U. Lin, Nikhil Wagle. Igf1r mediates cdk4/6 inhibitor (cdk4/6i) resistance in tumor samples and in cellular models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-08.

Abstract PS4-08: Biomarker analysis of paclitaxel, ganitumab, and metformin (PGM) therapy in the I-SPY2 neoadjuvant clinical trial

Abstract I-SPY2 is a neoadjuvant trial evaluating experimental therapies in combination with cytotoxic chemotherapy compared to chemotherapy alone with the primary endpoint of pathologic complete response (pCR). Abundant preclinical evidence suggested the type I insulin-like growth factor receptor (IGF-1R) regulated breast cancer growth, although multiple clinical trials did not show benefit. We were the first to report the results of a monoclonal IGF-1R antibody ganitumab (G) in combination with chemotherapy. PGM followed by doxorubicin/cyclophosphamide (AC) did not result in substantial increases in pCR when compared to P followed by AC. In this report, we examined several potential predictive biomarkers. IGF-1R inhibitors induce hyperglycemia and we examined hemoglobin A1C (HgbA1c) as a measure of glucose control in patients before and after PGM therapy. 106 patients received PGM and 104 patients had baseline HgbA1c with a median of 5.4%. However, 27% (28/104) had levels greater than 5.7% the upper limit of normal as defined by the NIDDK. 4 of 104 had HgbA1c greater than 6.5%, a level associated with type 2 diabetes. pCR rates are similar between patients with baseline HgbA1c ≤5.7% (21%) vs. >5.7% (25%) (Fisher test p=0.79). 72 of these patients had an additional HgbA1c during the course of PGM therapy. For patients with HgbA1c ≤5.7%, 27% (14/52) had subsequent elevation above 5.7% after PGM. For patients with a baseline HgbA1C >5.7%, all 20 patients continued to have elevated levels through PGM. We also examined pre-treatment tumor gene expression profiles derived from custom Agilent 44K full-genome microarrays. We studied 11 genes associated with the IGF-1R signaling (IGF1, IGF2, IGF1R, INSR, IGFBP2, IRS1, IRS2, IGFBP3, IGFBP4, IGFBP5, CDH1), the IGFBP5/IGFBP4 ratio, and two IGFR expression signatures (Creighton, et al. J Clin Oncol 26:4078 2008 PMID: 18757322; Mu, et al. Breast Cancer Res Treat 133:321 2012 PMID: 22297468). The 2 signatures evaluated: the IGF1 ligand score and the IGF1-R signature are anti-correlated (Rp= - 0.79). In the population as a whole, lower levels of IRS1 and IGFBP5 significantly associated with response to PGM (likelihood ratio test (LR) p< 0.05), as do lower levels of the IGF1 ligand score and higher levels of the IGF-1R signature. However, levels of IRS1 and the two expression signatures also trend toward or are significantly associated with response in the control arm; and treatment interactions for all four biomarkers are non-significant (LR p>0.05). Therefore, none of these biomarkers qualify as specific predictors of response to PGM. Similarly, high MammaPrint scores (MP2) were associated with higher pCR scores in both PGM and Control arms. Previous gene expression profiles were divided into tertiles (low, intermediate, high). Similar to the continuous case, IGF1Rsig-class associates with pCR in both the PGM and control arms (Fisher test p=0.033 and 0.044, respectively), and thus also fails as a specific predictor of response to PGM. We conclude that PGM therapy results in worsening of glucose control and likely increases serum insulin levels. While IGF gene expression profiling associated with treatment response, they were not specific for PGM. Further, biomarker analysis and strategies to control glucose will be needed to optimize anti-IGF-1R therapies. Citation Format: Douglas Yee, Paul Haluska, Denise M Wolf, Christina Yau, Amy Wilson, I-SPY2 TRIAL Consortium, Angela DeMichele, Claudine Isaacs, Jane Perlmutter, Joan Venticinque, Hope S Rugo, Richard Schwab, Nola M Hylton, W Fraser Symmans, Michelle E Melisko, Teresa L Helsten, Laura J van't Veer, Donald A Berry, Laura J Esserman. Biomarker analysis of paclitaxel, ganitumab, and metformin (PGM) therapy in the I-SPY2 neoadjuvant clinical trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-08.

Abstract 3055: Xentuzumab, a humanized IGF-1 and IGF-2 ligand co-neutralizing monoclonal antibody, shows efficacy in a human breast cancer model of bone metastasis

Abstract Bone metastases are a frequent complication of cancer and are associated with considerable morbidity. Bones provide a permissive environment for metastatic colonization due to their dynamic turnover and an abundance of secreted factors that are required for bone maintenance. Insulin-like growth factors (IGF) are the most abundant growth factors in bone and are required for normal skeletal development and function. IGFs also promote cancer progression, aggressiveness, and treatment resistance via activation of the IGF-1 receptor (IGF-1R) and insulin receptor variant A (IR-A). Of specific relevance to bone metastases, excessive bone destruction in osteolytic metastatic lesions leads to a release of IGF-1 from the bone matrix, thus promoting proliferation of metastatic tumor cells, stimulating tumor cell homing to bones, and contributing to further enhancement of bone destruction. Therapeutic targeting of the IGF axis may therefore provide an effective method for treating bone metastases. The aim of this study was to explore the efficacy of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[1]), alone and in combination with the mTOR inhibitor everolimus, in a breast cancer bone metastasis xenograft model. MDA-231-BoM cells[2], a subline of MDA-MB-231 selected in vivo for enhanced metastatic ability to bone, were injected intra-cardiacly to form bone metastases, and via tail vein to form lung/visceral metastases and treated with xentuzumab, everolimus, and the combination thereof. Tumor burden was monitored by bioluminescence imaging, and metastasis-associated osteolysis was measured by quantitative bone micro-computed tomography. Tumor-associated morbidity was evaluated by a multi-parameter health scoring scheme. Metastatic tumor burden in the bone was significantly reduced upon treatment with xentuzumab in monotherapy and in combination with everolimus vs. placebo. Xentuzumab (±everolimus) reduced the number and size of osteolytic lesions, delayed and diminished bone metastasis-associated morbidity, and prolonged survival. In contrast, the growth of lung/visceral metastases developing upon intravenous injection of tumor cells was not inhibited by xentuzumab. In summary, the IGF-1/-2 neutralizing antibody xentuzumab showed efficacy in a breast cancer bone metastasis model (with and without everolimus) but did not affect metastatic tumor growth in lung/visceral organs. A phase II trial evaluating the xentuzumab/ everolimus/ exemestane triple combination in HR+BC patients with non-visceral disease is currently ongoing (NCT03659136). [1] Friedbichler et al, 2014. Mol Cancer Ther 13(2):399 [2] Kang et al, 2003. Cancer Cell 3:537 Citation Format: Ulrike Weyer-Czernilofsky, Izabela Krecioch, Stephan Handschuh, Martin Glösmann, Mario Kuttke, Robin Jacob, Anna C. Obenauf. Xentuzumab, a humanized IGF-1 and IGF-2 ligand co-neutralizing monoclonal antibody, shows efficacy in a human breast cancer model of bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3055.

Abstract 527: Role of IGFBP3 in Neonatal Heart Regeneration

Background: The adult mammalian heart is unable to regenerate after an injury. However, newborn mice are able to fully regenerate the heart after myocardial infarction (MI). The neonatal MI model therefore is a potential blueprint for regenerating the adult heart that could offer novel therapies for patients suffering from heart disease. To investigate the mechanism by which neonatal heart regeneration occurs, we screened for secreted proteins that are upregulated after neonatal MI but not after adult MI. We hypothesized that such a protein could be a cardiomyocyte mitogen that underlies the cardiomyocyte proliferation that occurs after neonatal MI. Methods: We performed microarrays on neonatal and adult MI (and sham) heart tissue samples: we identified IGFBP3 (Insulin Growth Factor Binding Protein 3), which canonically transports IGF ligands in circulation, as a secreted protein that is uniquely upregulated after neonatal MI. We used in situ hybridization, reporter mice, and immunostaining to validate the findings from the microarray. Single cell RNA-seq data revealed that IGFBP3 is expressed in vascular cells. Results: We first tested whether IGFBP3 is necessary during neonatal heart regeneration: we performed neonatal (P1) MI in global Igfbp3 knockout mice and wild-type mice, and found that knockout mice have more fibrosis and worse ejection fraction (EF) one month after P1 MI. To determine if IGFBP3 is sufficient to promote cardiomyocyte proliferation, we injected recombinant IGFBP3 protein into the heart of one week-old mice (P7) and saw more cycling myocytes. We generated novel transgenic vascular-Igfbp3-overexpression mice, which exhibit less fibrosis as well as improved EF after P7 MI compared to controls. Conclusions: IGFBP3 is a secreted protein that is necessary for complete regeneration after a neonatal MI. Its ectopic expression can cause cardiomyocyte proliferation and can improve systolic function, and it prolongs the window of neonatal heart regeneration. Therefore, IGFBP3 may represent a cardiomyocyte mitogen with potential therapeutic value for adult heart disease.

Abstract B39: Modulation of insulin receptor alternative splicing to develop cancer therapeutics

Abstract Insulin receptor (IN-R) undergoes alternative splicing to produce two isoforms: the full-length IN-RB and exon 11 skipped IN-RA isoform. While IN-RB has high affinity for insulin and much lower affinity for IGF1 or IGF2 ligands, IN-RA binds both insulin and IGF2 with high affinity and as such it exploits the IGF pathway to accelerate the onset of tumor-cell hallmarks such as proliferation and angiogenesis. Our data show that there is a significantly increased expression of IN-RA levels in multiple cancer patient cohorts, including rhabdomyosarcoma (RMS) and osteosarcoma (OS). We also found this increased IN-RA expression in tumor cell lines derived from multiple different pediatric tumor types such as RMS, OS, and Ewing’s sarcoma as compared to the control samples. Additionally, our data show that cellular stress such as hypoxia increases the expression of the IN-RA isoform. Adaptation to hypoxic environments is a hallmark of the neoplastic phenotype, and pediatric sarcomas exhibit an intrinsically hypoxic physiology. However, how the INR alternatively spliced mRNAs are regulated is not well understood, nor is the role that these isoforms play in the initiation and progression and therapeutic resistance of human cancer. Our central hypothesis is that hypoxia alters the expression of splicing factors, leading to the generation of the IN-RA isoform, which contributes to cancer progression beyond the micrometastatic stage. We used an insulin receptor mini-gene system to identify a binding site for the RNA binding protein cugbp, critical for the hypoxia-induced splicing regulation of IN-R. We targeted the cugbp binding site using an antisense oligonucleotide (ASO) walk to identify an ASO that binds to the cugbp binding site in the intron 10 of INR gene and modulates the levels of endogenous IN-RA. To allow the possibility for therapeutic intervention, we utilized this ASO in OS cell lines. We found that the application of ASO shifts the IN-R splicing towards IN-RB and significantly reduces proliferation, migration, and angiogenesis in cancer cell lines. In order to delineate the downstream consequences of this splicing change, we subjected the control and ASO-treated cells to mass spectrometric analysis. Preliminary analysis of this data reveals that proliferation markers such as Ki67, PI3-kinase pathway components, protein clusters such as cell-cell adhesion, and cell division, among others, are significantly downregulated in cells treated with ASO where the splicing has been restored to the IN-RB isoform. Our data open a new paradigm of how alternative splicing regulates cell signaling to mediate cancer-causing changes, and our ASO compounds show promising insight into how we can reinstate the splicing pattern and impede tumorigenesis. Citation Format: Safiya Khurshid, Matias Montes, Brianne Sanford, Peter Houghton, Frank Rigo, Dawn Chandler. Modulation of insulin receptor alternative splicing to develop cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B39.

Microgravity, Bone Homeostasis, and Insulin-Like Growth Factor-1

Astronauts at are risk of losing 1.0–1.5% of their bone mass for every month they spend in space despite their adherence to high impact exercise training programs and diets high in nutrients, potassium, calcium, and vitamin D, all designed to preserve the skeletal system. This article reviews the basics of bone formation and resorption and details how exposure to microgravity or simulated microgravity affects the structure and function of osteoblasts, osteocytes, osteoclasts, and their mesenchymal and hematologic stem cell precursors. It details the critical roles that insulin-like growth factor-1 and its receptor insulin-like growth factor-1 receptor (GFR1) play in maintaining bone homeostasis and how exposure of bone cells to microgravity affects the function of these growth factors. Lastly, it discusses the potential of tumor necrosis factor-related apoptosis-inducing ligand, syncytin-A, sclerostin inhibitors and recombinant IGF-1 as a bone-saving treatment for astronauts in space and during their colonization of the Moon.

Abstract P5-03-05: Model of acquired resistance to the tyrphostin NT157 in hormone receptor-positive breast cancer

Abstract As previously reported, the small-molecule tyrphostin NT157 binds to the type I insulin-like growth factor-1 receptor (IGF-1R) beta subunit (IGF-IRβ) resulting in degradation of the insulin receptor substrate (IRS) adaptor proteins and inhibits growth of tamoxifen sensitive and resistant ER+ breast cancer cells (Yang, Y. et al. Horm Canc, in press). To investigate effects related to long-term NT157 exposure on IGF signaling, we incubated T47D parental cells in increasing concentrations of NT157 for 20 months. This prolonged exposure resulted in resistant cells (T47D-NTR). Monolayer growth assays showed parental cells inhibited at 0.7 μM of NT157 whereas resistant cells were suppressed by 8 μM of NT157. In parental cells, NT157 caused G0-G1 arrest and an increase in the sub-G1 fraction. However, resistant cells did not show these cell cycle changes after exposure to NT157. Furthermore, the antiproliferative effect of prolonged exposure to 96 hours of NT157 was correlated with decreased activation of Akt/S6k signaling axis and reduction of cyclin D1 expression in T47D parental cells. 48 hours treatment with 5 μM of NT157 resulted in a decrease of IGF-IR and insulin receptor (InR) expression level at 33% and 39% respectively, along with the downregulation of the adaptor proteins IRS-1/2 in only the parental cell line. In resistant cells, the NT157 treatment induced minimal IGF-1R suppression and a 21% increase of IR expression. Additionally, the T47D-NTR cells-maintained IRS protein levels at 10 μM. This absence of IRS protein degradation observed in NT157-resistant cells correlated with the continued expression of estrogen receptor-alpha (ERα), suggesting functional changes within IGF and ERα complex. IGF ligands (IGF-1 or IGF-2) and estradiol (E2) stimulated growth in both cell lines. When NT157 was removed from T47D-NTR cells, they were not re-sensitized to NT157 nor were they affected by IGF dependent growth in the re-introduction of NT157. These findings suggest that resistance to NT157 is mediated by the maintained expression of the IRS adaptor proteins. The continued responsiveness to estradiol and IGF ligands could be due to either a decrease in the drug's half-life or possible changes in NT157 binding to the beta subunit of IGF-IR in resistant cells. Citation Format: Ekyalongo RC, Abdelwahab R, Holtz A, Sabet A, Yee D. Model of acquired resistance to the tyrphostin NT157 in hormone receptor-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-05.