Sean C. Harrington

Transplantation of Circulating Endothelial Progenitor Cells Restores Endothelial Function of Denuded Rabbit Carotid Arteries

Background and Purpose— Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries. Methods— Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (105 cells in 200 μL saline) or 200 μL saline alone (control) were administered into the lumen of injured artery. Results— Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall. Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5, P<0.05). Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells. Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines. Conclusions— We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function. Paracrine effects of EPCs may contribute to regenerative properties of EPCs.

Genetic Deletion of Pregnancy-Associated Plasma Protein-A Is Associated With Resistance to Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice Challenged With a High-Fat Diet

Pregnancy-associated plasma protein–A (PAPP-A), a metalloproteinase in the insulin-like growth factor (IGF) system, is markedly upregulated in human atherosclerotic plaque. To determine whether PAPP-A plays an active role in the development of atherosclerosis, we crossed mice lacking apolipoprotein E (ApoE) with PAPP-A–deficient mice, generating ApoE knock-out (KO), PAPP-A KO, wild-type (WT/WT), and ApoE/PAPP-A double KO (KO/KO) mice. These mice were fed a high-fat diet starting at 7 weeks of age. Total serum cholesterol levels were elevated similarly in the ApoE KO and KO/KO mice and were 10-fold higher than in the WT/WT and PAPP-A KO mice. WT/WT and PAPP-A KO mice showed little or no lesion development even after 20 weeks of diet. ApoE KO mice had a progressive increase in aortic lesion area over 20 weeks of diet. In comparison, lesion area was reduced 60% to 80% in KO/KO mice. Lesions of ApoE KO aortas had 8- to 20-fold increases in PAPP-A, IGFBP-4, and IGF-I mRNA levels compared with nonlesional areas, whereas IGF-I receptor levels were equivalent—conditions for enhanced lesional IGF activity. Consistent with this, an in vivo marker of IGF-I receptor-mediated action was increased 10-fold in lesions from ApoE KO compared with KO/KO aortas. These data indicate that PAPP-A plays a critical role in lesion development in a mouse model of atherosclerosis, at least in part, through amplification of local IGF-I bioavailability.

Tumorgrafts as In Vivo Surrogates for Women with Ovarian Cancer

Purpose: Ovarian cancer has a high recurrence and mortality rate. A barrier to improved outcomes includes a lack of accurate models for preclinical testing of novel therapeutics. Experimental Design: Clinically relevant, patient-derived tumorgraft models were generated from sequential patients and the first 168 engrafted models are described. Fresh ovarian, primary peritoneal, and fallopian tube carcinomas were collected at the time of debulking surgery and injected intraperitoneally into severe combined immunodeficient mice. Results: Tumorgrafts demonstrated a 74% engraftment rate with microscopic fidelity of primary tumor characteristics. Low-passage tumorgrafts also showed comparable genomic aberrations with the corresponding primary tumor and exhibit gene set enrichment of multiple ovarian cancer molecular subtypes, similar to patient tumors. Importantly, each of these tumorgraft models is annotated with clinical data and for those that have been tested, response to platinum chemotherapy correlates with the source patient. Conclusions: Presented herein is the largest known living tumor bank of patient-derived, ovarian tumorgraft models that can be applied to the development of personalized cancer treatment. Clin Cancer Res; 20(5); 1288–97. ©2014 AACR.

Cell Biology of Pathologic Renal Calcification: Contribution of Crystal Transcytosis, Cell-Mediated Calcification, and Nanoparticles

Introduction The earliest lesion in the kidneys of idiopathic calcium oxalate stone formers is deposition of calcium phosphate in the interstitium, termed a Randalls plaque. Yet the cellular and molecular factors leading to their formation are unknown. Methods The influence of urinary proteins on adhesion of preformed calcium oxalate crystals to rat continuous inner medullary collecting duct (cIMCD) cells was studied in vitro, and cIMCD cells were also exposed to calcifying media containing β-glycerophosphate for up to 28 days. Renal tissue was obtained from a stone-forming and non-stone-forming individual at the time of nephrectomy. These nanoparticles, isolated from renal stones obtained at the time of surgical resection, were analyzed and propagated in standard cell culture medium. Results Urinary proteins influence crystal adhesion to renal epithelial cells, and this activity is abnormal in the urine of stone-forming patients. cIMCD cells assumed an osteoblastic phenotype when exposed to the calcifying medium, expressing two bone matrix proteins (osteopontin and bone sialoprotein) that were also identified in the kidney of the stone-forming patient and associated with crystal deposition. Nanoparticles were propagated from the majority of renal stones. Isolates were susceptible to selected metabolic inhibitors and antibiotics and contained conserved bacterial proteins and deoxyribonucleic acid (DNA). Conclusions These results suggest new paradigms for Randalls plaque formation and idiopathic calcium oxalate stone disease. It seems unlikely that these events are driven solely by physical chemistry; rather, they are critically influenced by specific proteins and cellular responses, and understanding these events will provide clues toward novel therapeutic targets.

Dual IGF-1R/InsR Inhibitor BMS-754807 Synergizes with Hormonal Agents in Treatment of Estrogen-Dependent Breast Cancer

Insulin-like growth factor (IGF) signaling has been implicated in the resistance to hormonal therapy in breast cancer. Using a model of postmenopausal, estrogen-dependent breast cancer, we investigated the antitumor effects of the dual IGF-1R/InsR tyrosine kinase inhibitor BMS-754807 alone and in combination with letrozole or tamoxifen. BMS-754807 exhibited antiproliferative effects in vitro that synergized strongly in combination with letrozole or 4-hydroxytamoxifen and fulvestrant. Similarly, combined treatment of BMS-754807 with either tamoxifen or letrozole in vivo elicited tumor regressions not achieved by single-agent therapy. Notably, hormonal therapy enhanced the inhibition of IGF-1R/InsR without major side effects in animals. Microarray expression analysis revealed downregulation of cell-cycle control and survival pathways and upregulation of erbB in response to BMS-754807 plus hormonal therapy, particularly tamoxifen. Overall, these results offer a preclinical proof-of-concept for BMS-754807 as an antitumor agent in combination with hormonal therapies in hormone-sensitive breast cancer. Cooperative cell-cycle arrest, decreased proliferation, and enhanced promotion of apoptosis may contribute to antitumor effects to be gauged in future clinical investigations justified by our findings.

Transgenic overexpression of pregnancy-associated plasma protein-A in murine arterial smooth muscle accelerates atherosclerotic lesion development

Pregnancy-associated plasma protein-A (PAPP-A) increases local IGF-I bioavailability through cleavage of inhibitory IGF binding protein (IGFBP)-4 in a variety of systems, including the cardiovascular system. To test the hypothesis that expression of PAPP-A promotes the development of atherosclerotic lesions, we generated transgenic mice that express human PAPP-A in arterial smooth muscle. Four founder lines were characterized for transgenic human PAPP-A mRNA and protein expression, IGFBP-4 protease activity, and tissue specificity. In study I, apolipoprotein E knockout (ApoE KO) mice, a well-characterized mouse model of atherosclerosis, and ApoE KO mice expressing the human PAPP-A transgene at relatively high levels (ApoE KO/Tg) were fed a high-fat diet. At harvest, aortas were dissected and opened longitudinally for en face staining of lipid-rich lesions. Lesion area was increased 3.5-fold in aortas from ApoE KO/Tg compared with ApoE KO mice (P < 0.001), but no significant difference was seen in lesion number. In study II, replacement of PAPP-A expression in arterial smooth muscle of double ApoE KO/PAPP-A KO mice resulted in a 2.5-fold increase in lesion area (P = 0.002), without an effect on lesion number. PAPP-A transgene expression was associated with a significant increase in an IGF-responsive gene (P < 0.001), suggesting increased local IGF-I action. We therefore conclude that expression of human PAPP-A localized to arterial smooth muscle accelerates lesion progression in a mouse model of atherosclerosis. These data provide further evidence for the importance of PAPP-A in the cardiovascular system and suggest PAPP-A as a potential therapeutic target in the control of atherosclerosis.

Shifts in the fecal microbiota associated with adenomatous polyps

Background: Adenomatous polyps are the most common precursor to colorectal cancer, the second leading cause of cancer-related death in the United States. We sought to learn more about early events of carcinogenesis by investigating shifts in the gut microbiota of patients with adenomas. Methods: We analyzed 16S rRNA gene sequences from the fecal microbiota of patients with adenomas (n = 233) and without (n = 547). Results: Multiple taxa were significantly more abundant in patients with adenomas, including Bilophila, Desulfovibrio, proinflammatory bacteria in the genus Mogibacterium, and multiple Bacteroidetes species. Patients without adenomas had greater abundances of Veillonella, Firmicutes (Order Clostridia), and Actinobacteria (family Bifidobacteriales). Our findings were consistent with previously reported shifts in the gut microbiota of colorectal cancer patients. Importantly, the altered adenoma profile is predicted to increase primary and secondary bile acid production, as well as starch, sucrose, lipid, and phenylpropanoid metabolism. Conclusions: These data hint that increased sugar, protein, and lipid metabolism along with increased bile acid production could promote a colonic environment that supports the growth of bile-tolerant microbes such as Bilophilia and Desulfovibrio. In turn, these microbes may produce genotoxic or inflammatory metabolites such as H2S and secondary bile acids, which could play a role in catalyzing adenoma development and eventually colorectal cancer. Impact: This study suggests a plausible biological mechanism to explain the links between shifts in the microbiota and colorectal cancer. This represents a first step toward resolving the complex interactions that shape the adenoma–carcinoma sequence of colorectal cancer and may facilitate personalized therapeutics focused on the microbiota. Cancer Epidemiol Biomarkers Prev; 26(1); 85–94. ©2016 AACR.

Quantifying insulin receptor isoform expression in FFPE breast tumors

BACKGROUND The development of predictive biomarkers for IGF targeted anti-cancer therapeutics remains a critical unmet need. The insulin receptor A isoform (InsR-A) has been identified as a possible biomarker candidate but quantification of InsR-A in widely available formalin fixed paraffin embedded (FFPE) tissues is complicated by its similarities with the metabolic signaling insulin receptor isoform B (InsR-B). In the present study, qPCR based assays specific for InsR-A, InsR-B and IGF-1R were developed for use in FFPE tissues and tested for feasible use in clinical archived FFPE estrogen receptor (ER)+and ER- breast cancer tumors. DESIGN FFPE compatible primer sets were designed with amplicon sizes of less than 60 base pairs and validated for target specificity, assay repeatability and amplification efficiency. FFPE tumors from ER+ (n=83) and ER-(n=64) primary untreated breast cancers, and ER+ hormone refractory (HR ER+) (n=61) breast cancers were identified for feasibility testing. The feasible use of InsR-A and InsR-B qPCRs were tested using all tumor groups and the feasibility of IGF-1R qPCR was determined using HR ER+ tumors. RESULTS All qPCR assays were highly reproducible with amplification efficiencies between 96-104% over a 6 log range with limits of detection of 4 or 5 copies per reaction. Greater than 90% of samples were successfully amplified using InsR-A, InsR-B or IGF-1R qPCR primer sets and greater than 88% of samples tested amplified both InsR isoforms or both isoforms and IGF-1R. InsR-A was the predominant isoform in 82% ER+, 68% ER- and 100% HR ER+ breast cancer. Exploratory analyses demonstrated significantly more InsR-A expression in ER+ and HR ER+ groups compared to InsR-B (ER+ p<0.05, HR ER+ p<0.0005) and both groups had greater InsR-A expression when compared to ER- tumors (ER+ p<0.0005, HR ER+ p<0.05). IGF-1R expression of HR ER+ tumors was lower than InsR-A (p<0.0005) but higher than InsR-B (p<0.0005). The InsR-B expression of HR ER+ tumors was significantly reduced compared other tumor subgroups (ER+ and ER-, p<0.0005) and lead to a significant elevation of HR ER+ InsR-A: InsR-B ratios (ER+ and ER-, p<0.0005). CONCLUSIONS The validated, highly sensitive InsR-A and InsR-B qPCR based assays presented here are the first to demonstrate the feasible amplification of InsR isoforms in FFPE tissues. Quantification data generated from this feasibility study indicating InsR-A is more predominant than InsR-B in breast cancer support the use of these assays for further investigation of InsR-A and InsR-B as predictive biomarkers for IGF targeted therapeutics.

IGFBP ratio confers resistance to IGF targeting and correlates with increased invasion and poor outcome in breast tumors

Purpose: To improve the significance of insulin-like growth factor–binding protein 5 (IGFBP-5) as a prognostic and potentially predictive marker in patients with breast cancer. Experimental Design: Increased IGFBP-5 expression was identified in MCF-7 cells resistant (MCF-7R4) to the IGF-1R/insulin receptor (InsR) inhibitor BMS-536924 and its role examined by targeted knockdown and overexpression in multiple experimental models. Protein expression of IGFBP-5 was measured by immunohistochemistry in a cohort of 76 patients with breast cancer to examine correlative associations with invasive tumor fraction and outcome. The use of a combined IGFBP-5/IGFBP-4 (BPR) expression ratio was applied to predict anti-IGF-1R/InsR response in a panel of breast cancer lines and outcome in multiple breast tumor cohorts. Results: IGFBP-5 knockdown decreased BMS-536924 resistance in MCF-7R4 cells, whereas IGFBP-5 overexpression in MCF-7 cells conferred resistance. When compared with pathologically normal reduction mammoplasty tissue, IGFBP-5 expression levels were upregulated in both invasive and histologically normal adjacent breast cancer tissue. In both univariate and multivariate modeling, metastasis-free survival, recurrence free survival (RFS), and overall survival (OS) were significantly associated with high IGFBP-5 expression. Prognostic power of IGFBP-5 was further increased with the addition of IGFBP-4 where tumors were ranked based upon IGFBP-5/IGFBP-4 expression ratio (BPR). Multiple breast cancer cohorts confirm that BPR (high vs. low) was a strong predictor of RFS and OS. Conclusion: IGFBP-5 expression is a marker of poor outcome in patients with breast cancer. An IGFBP-5/IGFBP-4 expression ratio may serve as a surrogate biomarker of IGF pathway activation and predict sensitivity to anti-IGF-1R targeting. Clin Cancer Res; 18(6); 1808–17. ©2012 AACR.

Complete IGF Signaling Blockade by the Dual-Kinase Inhibitor, BMS-754807, Is Sufficient To Overcome Tamoxifen and Letrozole Resistance In Vitro and In Vivo.

Resistance to hormonal therapy is a clinically unmet need in breast cancer. IGF signaling has been identified as a major mechanism of resistance to hormonal therapy in breast cancer. As components of the IGF signaling pathway are expressed in most breast cancers, the development of IGF-1R monoclonal antibody (mAb) and tyrosine kinase inhibitors (TKI) are active areas of clinical investigations. A key distinction between the mAb and TKIs are their differences in their ability to inhibit the Insulin Receptor (InsR). While targeting the InsR with TKIs may have a theoretical liability of hyperglycemia, targeting only the IGF-1R may have the theoretical liability of incompletely blocking IGF signaling. As InsR isoform A expression, which can transduce IGF-II-mediated proliferation, is higher in breast cancers compared to normal breast tissue, we investigated whether IGF-1R or IGF-1R/InsR inhibition was sufficient for overcoming resistance to hormonal therapy. To determine the optimal combination strategies for clinical investigations, we tested the hypothesis that IGF signaling inhibition could overcome primary (or de novo/intrinsic) and secondary (or acquired/selected) resistance to hormonal therapy. For these studies, we used either hormone therapy-naive or hormone therapy-resistant variants of the breast cancer model, MCF-7/AC-1, which has been engineered to stably express full-length human aromatase. We employed and compared a novel, potent dual kinase inhibitor of the IGF-1R and InsR, BMS-754807, which is currently in early clinical investigations, with the IGF-1R antibody mAb391. BMS-754807 has been shown to induce apoptosis more potently than mAb391 in Rh41 human rhabdomyosarcoma cells. In vitro, BMS-754807 demonstrated profound synergy in combination with tamoxifen and letrozole (median effect combination index In vivo , BMS-754807 enhanced the anti-tumor activity of tamoxifen and letrozole in hormone-naive tumors and induced regression of tumors resistant to tamoxifen or letrozole when combined with letrozole. This activity was not observed with mAb therapy, which resulted in greater up-regulation of InsR-A and erbB receptor expression and activation. This suggested a greater susceptibility to resistance pathways with mAb therapy. Dual IGF-1R/InsR blockade alone or in combination was tolerated by the animals and has no significant change in glucose homeostasis. Gene expression profiling experiments to compare the difference between the effects of tamoxifen in combination with BMS-754807 and with mAb revealed alternative pathway signaling is one of the potential mechanisms of resistance.In summary, combined hormonal therapy with BMS-754807 overcomes primary and secondary resistance to tamoxifen and letrozole and was well tolerated. IGF-1R blockade with a mAb alone is insufficient to overcome resistance and induces InsR over-expression. Thus, IGF signaling through either InsR or IGF-1R may be a major mechanism of resistance to hormonal therapy. These data suggest that blockade of IGF-1 and IGF-II from activation of IGF-1R and InsR, with agents such as BMS-754807 have promise in extending the benefits of hormonal therapy in breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 402.