Jurriaan Brouwer-Visser

IGF2 signaling and regulation in cancer

Upregulation of IGF2 occurs in both childhood and adult malignancies. Its overexpression is associated with resistance to chemotherapy and worse prognosis. IGF2 promoter usage is developmentally regulated; however, malignant tissues are characterized by re-activation of the fetal IGF2 promoters, especially P3. In this review, we describe the mechanisms of IGF2 signaling and regulation in normal and malignant tissues and their clinical implications.

Insulin-Like Growth Factor 2 Silencing Restores Taxol Sensitivity in Drug Resistant Ovarian Cancer

Drug resistance is an obstacle to the effective treatment of ovarian cancer. We and others have shown that the insulin-like growth factor (IGF) signaling pathway is a novel potential target to overcome drug resistance. The purpose of this study was to validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and to determine the efficacy of targeting IGF2 in vivo. An analysis of The Cancer Genome Atlas (TCGA) data in the serous ovarian cancer cohort showed that high IGF2 mRNA expression is significantly associated with shortened interval to disease progression and death, clinical indicators of drug resistance. In a genetically diverse panel of ovarian cancer cell lines, the IGF2 mRNA levels measured in cell lines resistant to various microtubule-stabilizing agents including Taxol were found to be significantly elevated compared to the drug sensitive cell lines. The effect of IGF2 knockdown on Taxol resistance was investigated in vitro and in vivo. Transient IGF2 knockdown significantly sensitized drug resistant cells to Taxol treatment. A Taxol-resistant ovarian cancer xenograft model, developed from HEY-T30 cells, exhibited extreme drug resistance, wherein the maximal tolerated dose of Taxol did not delay tumor growth in mice. Blocking the IGF1R (a transmembrane receptor that transmits signals from IGF1 and IGF2) using a monoclonal antibody did not alter the response to Taxol. However, stable IGF2 knockdown using short-hairpin RNA in HEY-T30 effectively restored Taxol sensitivity. These findings validate IGF2 as a potential therapeutic target in drug resistant ovarian cancer and show that directly targeting IGF2 may be a preferable strategy compared with targeting IGF1R alone.

Insulin/IGF and sex hormone axes in human endometrium and associations with endometrial cancer risk factors

PurposeExperimental and observational data link insulin, insulin-like growth factor (IGF), and estrogens to endometrial tumorigenesis. However, there are limited data regarding insulin/IGF and sex hormone axes protein and gene expression in normal endometrial tissues, and very few studies have examined the impact of endometrial cancer risk factors on endometrial tissue biology.MethodsWe evaluated endometrial tissues from 77 premenopausal and 30 postmenopausal women who underwent hysterectomy for benign indications and had provided epidemiological data. Endometrial tissue mRNA and protein levels were measured using quantitative real-time PCR and immunohistochemistry, respectively.ResultsIn postmenopausal women, we observed higher levels of phosphorylated IGF-I/insulin receptor (pIGF1R/pIR) in diabetic versus non-diabetic women (p value =0.02), while women who reported regular nonsteroidal anti-inflammatory drug use versus no use had higher levels of insulin and progesterone receptors (both p values ≤0.03). We also noted differences in pIGF1R/pIR staining with OC use (postmenopausal women only), and the proportion of estrogen receptor-positive tissues varied by the number of live births and PTEN status (premenopausal only) (p values ≤0.04). Compared to premenopausal proliferative phase women, postmenopausal women exhibited lower mRNA levels of IGF1, but higher IGFBP1 and IGFBP3 expression (all p values ≤0.004), and higher protein levels of the receptors for estrogen, insulin, and IGF-I (all p values ≤0.02). Conversely, pIGF1R/pIR levels were higher in premenopausal proliferative phase versus postmenopausal endometrium (p value =0.01).ConclusionsThese results highlight links between endometrial cancer risk factors and mechanistic factors that may contribute to early events in the multistage process of endometrial carcinogenesis.

RNA-seq Identification of RACGAP1 as a Metastatic Driver in Uterine Carcinosarcoma.

Purpose: Uterine carcinosarcoma is a rare aggressive malignancy frequently presenting at advanced stage of disease with extrauterine metastases. Median survival is less than 2 years due to high relapse rates after surgery and poor response to chemotherapy or radiotherapy. The goal of this study was to identify novel therapeutic targets. Experimental Design: We applied RNA-seq analysis to prospectively collected uterine carcinosarcoma tumor samples from patients undergoing primary surgical resection and for comparison, normal endometrial tissues from postmenopausal women undergoing hysterectomy for benign indications. Functional assays were done in primary carcinosarcoma cell lines developed from patients and in established cell lines, as well as a cell line–derived xenograft model. Validation was done by analysis of an independent cohort of patients with uterine carcinosarcoma from The Cancer Genome Atlas (TCGA). Results: Rac GTPase–activating protein 1 (RACGAP1) was identified to be highly upregulated in uterine carcinosarcoma. Functional assays showed that RACGAP1 mediates motility and invasion via regulation of STAT3 phosphorylation and survivin expression. RACGAP1 depletion or survivin inhibition abrogated motility and invasiveness of carcinosarcoma cells, while RACGAP1 overexpression conferred invasiveness to endometrial adenocarcinoma cells. In the TCGA cohort, RACGAP1 expression correlated with survivin expression and extrauterine spread of disease. Conclusions: The RACGAP1–STAT3–survivin signaling pathway is required for the invasive phenotype of uterine carcinosarcoma and is a newly identified therapeutic target in this lethal disease. Clin Cancer Res; 22(18); 4676–86. ©2016 AACR.

Abstract 1080: Effect of IGF2 overexpression on the tumorigenicity of human ovarian carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction High Insulin-like Growth Factor 2 (IGF2) tumor expression correlates with reduced disease-free survival in epithelial ovarian tumors. IGF2 mRNA is also upregulated after Taxol treatment of ovarian cancer cells. The purpose of this study was to determine the effect of IGF2 overexpression on the tumorigenicity of human ovarian cancer cells. Methods HEY cells were stably transfected using lentiviral particles with IGF2 or no insert (EV) constructs. qPCR and ELISA were performed to measure the IGF2 mRNA and secreted protein expression levels, respectively. Signaling pathways were probed by immunoblotting. Tumorigenicity was evaluated in athymic nude mice by subcutaneous injection of serial cell dilutions with matrigel, followed by monitoring of in vivo tumor growth by caliper measurements. qPCR for IGF2 mRNA and immunohistochemistry for Ki67 were performed on the excised tumors. Results The IGF2 mRNA level was 4679 ±176 fold-change higher in HEY-IGF2 cells relative to HEY-EV cells (P<0.0001). The amount of IGF2 protein secreted in 48 h was 5159 ng/100,000 HEY-IGF2 cells (51.6 pg/cell), and undetectable in the media of HEY-EV cells. Tyrosine phosphorylation of the IGF1-receptor was increased 13-fold in HEY-IGF2 cells. In vitro, dependency on the IGF1-receptor signaling was unaltered, as IGF2 overexpressing cells responded similarly to the IGF1-receptor inhibitor NVP-AEW541 compared to HEY-EV cells. Following an injection of one million cells with matrigel, the mean tumor volume of xenografts after 4 weeks was 1608 ±330 mm3 versus 705 ±95 mm3 for HEY-IGF2 and HEY-EV xenografts, respectively (P=0.04, N=6 per group). Immunohistochemistry for Ki67 showed a significantly higher percentage of stained nuclei in HEY-IGF2 tumors than in HEY-EV tumors (P=0.04, N=3 per group). Discussion Stable overexpression of IGF2 in HEY ovarian cancer cells resulted in an increased xenograft growth rate. Further evaluation is underway to fully characterize the mechanisms by which IGF2 overexpression promotes tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1080. doi:1538-7445.AM2012-1080

Abstract 948: In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer.

Introduction. We previously showed that paclitaxel-resistance of human ovarian carcinoma cell lines was reverted by knockdown of IGF2 with RNA interference. The aim of this study was to determine if our paclitaxel-resistant cell line HEY-T30 was resistant to paclitaxel treatment in vivo and if this resistance could be reverted by IGF2 knockdown through short hairpin RNA. Methods. Paclitaxel-resistant HEY-T30 cells, derived in our laboratory from HEY ovarian carcinoma cells, were transfected with a plasmid containing shRNA to target IGF2 (shIGF2-p) or a nontargeting shRNA (shScrambled). Stably transfected clones were evaluated by reverse transcription real-time PCR for IGF2 mRNA expression and by sulforhodamine cytotoxicity assay for paclitaxel sensitivity. For in vivo experiments, HEY, HEY-T30, shIGF2-p or shScrambled cells were suspended in OptiMem and one million cells/animal injected subcutaneously into 4-week-old female nude mice. Tumors were allowed to grow to a mean volume of 125 mm3, prior to treatment initiation with paclitaxel (20 mg/kg administered intraperitoneally every 3 days x 5 doses) or vehicle (5% dextrose in water; D5W). Animals were weighed and tumors were measured with digital calipers three times per week. Effect of treatment was calculated as effect=1-(T/C) at the indicated time point, where T is the mean tumor volume of the paclitaxel treatment group and C is the mean tumor volume of the vehicle control group. Significance was calculated with a 2-way repeated measures ANOVA with a Bonferroni multiple comparisons post-test. Results. IGF2 mRNA levels of shIGF2-p cells were comparable to HEY, whereas shScrambled was similar to HEY-T30. The IC50s (concentration of 50% inhibition of proliferation) for paclitaxel in vitro were HEY: 2.3 nM; HEY-T30: 164.0 nM; shIGF2-p: 13.89 nM; shScrambled: 140.0 nM. HEY xenografts responded well to paclitaxel treatment, showing a significant growth retardation compared to the D5W group beginning at 9 days after treatment initiation (p=0.0005), with an effect of treatment of 83.2% at day 19. In contrast, HEY-T30 xenografts showed no significant difference in tumor volume between the paclitaxel-treated and the D5W group at any time point, confirming its resistance to paclitaxel (the effect of treatment was 5.5%). shScrambled xenografts were similarly resistant to paclitaxel as HEY-T30 xenografts. shIGF2-p xenografts responded well to paclitaxel treatment, showing a difference in tumor volume from day 15 onward (day 15, p=0.0457; day 19, p=0.0006) with an effect of treatment of 64.1% after 22 days. Discussion. HEY-T30 xenografts are resistant to paclitaxel treatment, whereas HEY xenografts respond well. IGF2 knockdown by shRNA restores paclitaxel sensitivity to the HEY-T30 xenografts. These in vivo results confirm our prior observations in cell lines and suggest a novel target for the treatment of paclitaxel-resistant disease. Citation Format: Jurriaan Brouwer-Visser, Jiyeon Lee, Maria J. Cossio, Gloria S. Huang. In vivo evaluation of IGF2 RNA interference in a mouse xenograft model of paclitaxel-resistant human ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 948. doi:10.1158/1538-7445.AM2013-948

Abstract A48: Effect of constitutive IGF2 overexpression on tumorigenicity of human ovarian carcinoma cells

Introduction: We previously described the association of high tumoral IGF2 expression with reduced disease-free survival in epithelial ovarian tumors. The purpose of this study was to determine the effect of IGF2 overexpression on the tumorigenicity of human ovarian cancer cells. Methods: Expression constructs containing the full-length coding sequence of IGF2 or no insert (empty vector; EV) were made. HEY cells were stably transfected. Quantitative real-time PCR (of CDNA made from cell and xenograft RNA) and ELISA (of conditioned media) were performed to measure the IGF2 mRNA and protein expression levels, respectively. The relative IGF2 mRNA expression level was calculated by the 2 −δδCt method with internal normalization to the cyclophilin B mRNA level. Tumorigenicity was evaluated in athymic nude mice by subcutaneous injection of 1×10 6 cells, with or without matrigel, followed by serial monitoring of in vivo tumor growth by digital caliper measurements. The two-tailed t-test was used to evaluate the significance of comparisons. Results: The IGF2 mRNA level was 5604 ±617 fold-change higher in HEY-IGF2 cells relative to HEY-EV cells (P 3 versus 52 ±15 mm 3 for HEY-IGF2 and HEY-EV, respectively (P 3 versus 705 ±95 mm 3 for HEY-IGF2 and HEY-EV xenografts, respectively (P Conclusions: Stable overexpression of IGF2 in HEY ovarian cancer cells and xenografts was achieved, and resulted in an increase in IGF2 protein secretion. IGF2 overexpression enhanced in vivo tumorigenicity of HEY ovarian cells, accompanied by an increased xenograft growth rate. Further evaluation is underway to fully characterize the mechanisms by which IGF2 overexpression promotes tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A48.

Abstract 1228: Resistance to discodermolide, a microtubule stabilizing agent and senescence inducer, is 4E-BP1 dependent

Discodermolide is a microtubule stabilizing agent that induces accelerated cell senescence. A discodermolide-resistant cell line, AD32, was generated from the human lung cancer cell line A549. We hypothesize that the major resistance mechanism in these cells is escape from accelerated senescence. AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells. Lentiviral-mediated re-expression of wild type 4E-BP1 in AD32 cells increased the proliferation rate and reverted resistance to discodermolide via restoration of discodermolide-induced accelerated senescence. Consistent with this, cell growth and response to discodermolide was confirmed in vivo using tumor xenograft models. Furthermore, re-introduction of a non-phosphorylatable mutant (Thr 37/46 Ala) of 4E-BP1 was able to partially restore sensitivity and enhance proliferation in AD32 cells, suggesting that these effects are independent of phosphorylation by mTORC1. Microarray profiling of AD32 resistant cells versus sensitive A549 cells, and subsequent unbiased gene ontology analysis, identified molecular pathways and functional groupings of differentially expressed mRNAs implicated in overcoming discodermolide-induced senescence. The most-statistically significant classes of differentially expressed genes included p53 signaling, G2/M checkpoint regulation and genes involved in the role of BRCA1 in the DNA damage response. Consistent with this, p53 protein expression was up-regulated and had increased nuclear localization in AD32 cells relative to parental A549 cells. Furthermore, the stability of p53 was enhanced in AD32 cells. Our studies propose a role for 4E-BP1 as a regulator of discodermolide-induced accelerated senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1228. doi:10.1158/1538-7445.AM2011-1228

Abstract A33: in ovarian cancer cell lines resistant to microtubule-stabilizing agents, expression of IGF2 and ABCB1 is increased and IGF1R inhibition restores drug sensitivity

Overexpression of insulin-like growth factor 2 (IGF2) is associated with worse outcome in ovarian cancer patients; previously, our group has shown that IGF2 is upregulated after Taxol treatment of ovarian cancer cells and leads to activation of AKT. Thus, we hypothesized that IGF2 may be involved in drug resistance. The Taxol-resistant cell line HEY-T30 was developed in our laboratory from chemo-naive, sensitive HEY ovarian cancer cells by repeated Taxol treatment in tissue culture. HEY-T30 is 40-fold resistant to Taxol (IC50: 2.6 nM versus 105.9 nM), as determined by sulforhodamine B cytotoxicity assays. In these cells, IGF2 mRNA levels, measured by quantitative PCR, are increased 8.8-fold compared with its expression in the parental cell line. In addition, HEY-T30 have a genomic amplification of ABCB1 (mdr1; encoding the drug efflux pump p-glycoprotein) detected by array CGH, associated with increased ABCB1 mRNA expression. Alterations in IGF2 and ABCB1 were assessed in an additional drug-resistant ovarian cancer cell line, OVCAR8-D30, developed in our laboratory. OVCAR8-D30 is 2-fold-resistant to discodermolide (IC50: 56 nM versus 115 nM), another microtubule-stabilizing drug with a distinct chemical structure. This resistant cell line also showed an increase (4-fold) in IGF2 expression compared to its parental cell line, as well as a 19-fold increase in ABCB1 expression. To determine the effect of IGF pathway inhibition in Taxol-resistant cells, NVP-AEW541 (a small molecule inhibitor of the IGF1R, the major receptor for IGF2) was used. By concurrent treatment with NVP-AEW541, HEY-T30 cells were significantly re-sensitized to Taxol (IC50 from 105.9 nM to 17.0 nM). The effect of combined Taxol and NVP-AEW541 treatment on cell cycle distribution was evaluated by flow cytometry. Eighteen hours after Taxol (75 nM, 125 nM and 250 nM) and NVP-AEW541 (1 μM) treatment alone or in combination, more cells were arrested in G2/M with combination treatment compared to Taxol treatment alone at the higher Taxol concentrations (75 nM: 23.3% to 26.4% with NVP, 125 nM: 23.8% to 44.0% with NVP, and 250 nM: 34.6% to 74.1% with NVP) as determined by flow cytometry. At this time point, caspase activity analysis by flow cytometry detection of FAM-VAD-FMK FLICA reagent, showed no significant differences between the two treatments (75 nM: 6.7% and 9.5% with NVP, 125 nM: 10.0% and 11.5% with NVP, and 250 nM: 12.4% and 14.9% with NVP). These data suggest that treatment with the IGF1R inhibitor NVP-AEW541 can restore Taxol sensitivity in cells with multiple aberrations that confer drug-resistance (IGF2 and ABCB1 over-expression). We conclude that IGF2 has a prominent role in resistance to microtubule-stabilizing drugs in ovarian cancer cell lines and we therefore propose IGF2 to be a valuable target in future molecular therapies for chemo-resistant ovarian cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A33

Abstract 1809: Reversal of Taxol resistance in ovarian cancer cell lines by IGF pathway inhibition

Our group recently reported increased insulin-like growth factor 2 (IGF2) expression and AKT activation in ovarian cancer cells following treatment with the microtubule-stabilizing agent Taxol. We hypothesized that IGF2 is a modulator of Taxol resistance. Therefore, we developed cell line models of Taxol-resistance in A2780 and HEY, two ovarian cell lines, by repeated treatment with increasing Taxol concentrations, with or without concurrent verapamil, a p-glycoprotein inhibitor. The involvement of IGF2 in drug resistance was evaluated by real time PCR, immunoblotting, siRNA knockdown of IGF2, and small-molecule inhibition of the IGF1 Receptor (IGF1R; which is the major receptor for IGF2). In both of the resistant cell lines (A2780-Tx8Vp, 3-fold Taxol-resistance; and HEY-T30, 40-fold Taxol-resistance), IGF2 mRNA expression, measured by real time PCR, was significantly elevated compared with the parental cell lines, while IGF1R expression, assessed by immunoblotting, was mildly decreased. IGF1R inhibition by the small molecule NVP-AEW541 dramatically re-sensitized both resistant cell lines to Taxol, and cell cycle analysis demonstrated that concurrent NVP-AEW541 and Taxol significantly enhanced G2/M arrest, compared with Taxol alone. While A2780-Tx8Vp cells did not express detectable mdr1/abcb1 (encoding p-glycoprotein), HEY-T30 cells did over-express mdr1 (and were sensitized to Taxol treatment by verapamil). To exclude a possible interaction with p-glycoprotein, we evaluated IGF2 siRNA as an alternate, more specific approach of targeting the IGF pathway in HEY-T30 cells. It was found that IGF2 depletion by siRNA significantly enhanced the antiproliferative effect of Taxol in these cells. In summary, IGF2 modulates Taxol resistance in these ovarian cancer cell lines. IGF pathway inhibition, by IGF2 depletion or by inhibition of its major receptor IGF1R, reverses Taxol resistance in these cells. Such novel findings suggest that IGF2 could be a therapeutic target for ovarian cancer, particularly in the setting of Taxol resistance. 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 1809.