Maria J. Cossio

REST-dependent epigenetic remodeling promotes the developmental switch in synaptic NMDA receptors

NMDA receptors (NMDARs) are critical to synaptogenesis, neural circuitry and higher cognitive functions. A hallmark feature of NMDARs is an early postnatal developmental switch from those containing primarily GluN2B to primarily GluN2A subunits. Although the switch in phenotype has been an area of intense interest for two decades, the mechanisms that trigger it and the link between experience and the switch are unclear. Here we show a new role for the transcriptional repressor REST in the developmental switch of synaptic NMDARs. REST is activated at a critical window of time and acts via epigenetic remodeling to repress Grin2b expression and alter NMDAR properties at rat hippocampal synapses. Knockdown of REST in vivo prevented the decline in GluN2B and developmental switch in NMDARs. Maternal deprivation impaired REST activation and acquisition of the mature NMDAR phenotype. Thus, REST is essential for experience-dependent fine-tuning of genes involved in synaptic plasticity.

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.

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

Insulin-like growth factor 2: a poor prognostic biomarker linked to racial disparity in women with uterine carcinosarcoma

The objective of this study was to investigate the relationship of insulin‐like growth factor 2 (IGF2) expression and survival in women with uterine carcinosarcoma (UCS). Insulin‐like growth factor 2 protein expression was determined by immunohistochemical staining of tumor tissues from 103 patients with UCS. The H‐score (product of staining intensity and percentage positive cells) was quantified for the epithelial cytoplasmic (EC), epithelial nuclear (EN), and malignant stromal compartments. Multivariable Cox proportional hazard regression models were used to examine the relationship of IGF2 levels with progression‐free survival (PFS) and overall survival (OS). Adjusting for stage, race, and adjuvant therapy, PFS and OS were reduced in patients with high IGF2 (H‐score ≥ median) in the EC and EN compartments. Black race was independently associated with reduced PFS and OS in patients with early‐stage disease, and IGF2 levels in the EC were higher in black than in white patients (P = 0.02, Wilcoxon test). In a race‐stratified multivariable analysis, high IGF2 in the epithelial compartments more than doubled the risk of death in black women; HR = 2.43 (95% CI: 1.18–5.01, P = 0.02) for high IGF2 in the EC; and HR = 2.34 (95% CI: 1.25–4.39, P = 0.008) for high IGF2 in the EN. In conclusion, high tumor IGF2 expression is an independent risk factor for reduced PFS and OS in UCS. Black women have elevated tumor IGF2 compared with white women, and decreased survival associated with high IGF2. These findings identify IGF2 as a candidate biomarker for survival linked to racial disparity in women with UCS.

Transforming growth factor β1 and extracellular matrix protease expression in the uterosacral ligaments of patients with and without pelvic organ prolapse.

Objectives This study was undertaken to evaluate the expression of transforming growth factor &bgr;1 (TGF-&bgr;1) and matrix metalloproteinase 9 (MMP-9), key regulators of the extracellular matrix composition, in the uterosacral ligaments (USLs) of women with pelvic organ prolapse (POP) compared with controls. Methods Under an institutional review board approval, USL samples were obtained from women undergoing vaginal hysterectomy for stage 2 or greater POP (cases, n = 21) and from women without POP undergoing vaginal hysterectomy for benign indications (controls, n = 19). Hematoxylin and eosin and trichrome staining were performed on the USL sections, and the distribution of smooth muscle and fibrous tissue were quantified. Immunohistochemical staining was performed using anti–TGF-&bgr;1 and anti–MMP-9 antibodies. The expressions of TGF-&bgr;1 and MMP-9 were evaluated by the pathologist, who was blinded to all clinical data. Results Transforming growth factor &bgr;1 expression positively correlated with MMP-9 expression (R = 0.4, P = 0.01). The expressions of TGF-&bgr;1 and MMP-9 were similar in subjects with POP versus controls. There was a significant increase in fibrous tissue (P = 0.008) and a corresponding decrease in smooth muscle (P = 0.03), associated with increasing age. The TGF-&bgr;1 expression, but not MMP-9 expression, also significantly increased with age (P = 0.02). Discussion Although our study uncovered age-related alterations in USL composition and TGF-&bgr;1 expression, there was no difference in the expression of TGF-&bgr;1 or MMP-9 in the subjects with POP versus controls.

Abstract 5131: Establishment of uterine carcinosarcoma primary cell lines for chemosensitivity testing and evaluation of targeted therapy

To establish primary cell lines from uterine carcinosarcoma (CS) patient samples and to evaluate combination treatment with standard chemotherapy with or without the addition of an anti-IGF1R antibody. CS tumor tissue was obtained under IRB approval at the time of primary surgery. We reported the establishment of patient-derived xenografts elsewhere. Tissue pieces were minced extensively and digested by collagenase treatment. The single cell solution was then seeded in F-media containing EGF, insulin, hydrocortisone, adenine, cholera toxin and the Rock inhibitor Y-27632. A previously established carcinosarcoma cell line CS99 (Schulten et al, 2008) was used for comparison. To verify origin, Short Tandem Repeat (STR) profiles of the primary cell lines CS13, CS18, CS19, CS21 and CS22 were compared with the patient9s tumor samples. Drug sensitivity to Taxol, cisplatin and carboplatin was determined individually or in combination using the sulforhodamine B (SRB) proliferation assay. The effect of IMC-A12 (anti-IGF1R antibody, Eli Lily / Imclone) alone or with chemotherapy was also determined with the SRB assay. Total and promoter-specific IGF2 mRNA levels were determined by reverse transcriptase quantitative PCR. All STR profiles of the primary cell lines matched their patient sample counterparts. IGF2 mRNA expression levels of the cell lines were similar to the patient samples, and over 10,000-fold higher than in CS99. IGF2 promoter-specific primers showed that the IGF2 mRNA transcripts of the primary cell lines were initiated at the oncofetal IGF2 promoters P3 and P4. For Taxol, the IC50 (inhibitory concentration of 50%) ranged from 4.7 nM for CS21 to 9.8 nM for CS22. For cisplatin, the IC50 ranged from 1.0 μM for CS99 to 4.8 μM for CS19. For carboplatin, CS99 cells had the lowest IC50 of 12.5 μM while CS19 had the highest IC50 of 58.1 μM. In the presence of 10 μg/ml IMC-A12, the primary cell lines CS19, CS21 and CS22 showed significant sensitization to Taxol (∼50% decrease in IC50), to carboplatin (∼65% decrease in IC50), and to combination Taxol/carboplatin treatment (∼50% decrease in IC50). We conclude that establishing primary cell lines of this rare cancer is a promising approach for testing sensitivity to current and novel chemotherapies and combination treatment strategies. We also found that high oncofetal promoter-driven IGF2 mRNA expression was observed in all CS samples tested and that IGF1R blockade sensitized the primary cell lines to standard chemotherapy. Citation Format: Jurriaan Brouwer-Visser, Eirwen Scott, Shijun Mi, Maria J. Cossio, Tiffany Hebert, Gloria S. Huang. Establishment of uterine carcinosarcoma primary cell lines for chemosensitivity testing and evaluation of targeted therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5131. doi:10.1158/1538-7445.AM2015-5131

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