Dr. GAGAN DUDEJA

Dr. THIRUMALESH M. B.

Semi Finals

Abstract

Purpose: To identify EMT expression profile in retinoblastoma subjects & develop in-vitro model to elucidate the signalling mechanisms underlying vitreous seeding and metastasis.

Methods: Microarray was performed on Rb tumor samples (n=9) & paediatric control retina (n=2) to identify EMT specific genes. Q-PCR & western blot was performed on WERI-Rb1 cells, under RB1 null & RB1 overexpression, to assess EMT related genes & protein expression.

Results: Differential expression of key EMT markers like Twist, E-cadherin, N-cadherin & Vimentin (p≤0.05, FC ≥ 2.0) were identified using microarray. In WERI-Rb1 cells, RB1 overexpression displayed significant reduction of EMT markers like Vimentin, Twist, N-cadherin, while E-cadherin displayed an increase in protein expression. RB1 overexpression also displayed significant reduction in phospho β-catenin expression at the protein level.

Conclusion: RB1 dependent EMT repression mechanism can halt metastatic propensity & therapeutic resistance.

Full Text

Introduction:
Retinoblastoma (Rb) is an aggressive pediatric ocular malignancy that manifest covertly with leukocoria and threatens the survival of patients. Rb is predisposed primarily by genetic mutations in RB1 in one or more cells of the retina[1]. It may be triggered by the loss of function or mechanistic alterations of other genes by various chromosomal or mutational events [2]. The incidence of retinoblastoma is 1 in 18,000 live births[2], with about estimated 2000 children being diagnosed in India each year[3]. The tumor appears as a unifocal or multifocal yellow-white retinal mass with a feeding retinal artery and draining vein. There is often surrounding subretinal fluid, subretinal seeds, or vitreous seeds [4]. Management of a child with retinoblastoma involves a balance of patient life with globe salvage and ultimate visual potential. The current management strategies has evolved from enucleation, brachytherapy, focal therapy, cryotherapy, transpupillary thermotherapy, laser photocoagulation to advanced chemotherapy comprising of various delivery routes and chemotherapy protocols [5, 6]. However, enucleation continues to remain as critical choice for advanced retinoblastoma with metastatic phenotype, particularly in Asia and Africa [7].

Advanced Rb tumors demonstrate massive choroidal invasion [8] and metastatic spread, primarily through optic nerve [9] and sclera [10], to regional lymph nodes, central nervous system (CNS) and bone marrow[11] causing potent threat to vision and life. Clinically, to manage metastatic Rb tumors, a very intensive multimodality approach incorporating high dose chemotherapy regimens involving carboplatin, etoposide and cyclophosphamide followed by radiation and autologous stem cell therapy are currently being considered [12]. However, advanced tumors evolve during each chemotherapy cycle and develop resistance to anticancer therapeutics, diminishing the efforts of the clinical management procedures [13, 14]. Metastatic tumors acquire chemotherapy resistance through trans-differentiation initiated by the epithelial to mesenchymal transition (EMT) program in different cancers [15] [16]. EMT program begins with the loss of epithelial phenotypes by downregulation of E-cadherin and tight junction adhesion molecules. The differentiated cancer cells transit to mesenchymal phenotype with an invasive dedifferentiated characteristic, eventually acquiring chemo-drug resistance.

In the present study, we discovered key EMT genes that drive metastatic dissemination in advanced Rb tumors. We profiled EMT signatures using microarray in advanced and non advanced Rb tumors and found key mesenchymal transition factors like ZEB1, Twist & N-cadherin to be significantly upregulated in advanced Rb tumors. Using in-vitro systems, we further investigated the underlying mechanisms that trigger EMT genes to acquire metastatic phenotype in advanced Rb tumors.

Methods:
Clinical samples
The study was conducted in accordance with the Declaration of Helsinki principles under a protocol approved by institutional ethics committee of Narayana Nethralaya (EC Ref no: C/2013/03/02). Informed written consents were received from all parents before inclusion in the study. Histology confirmed Rb tumors (n=9) comprising of Group E and Group D of age range 0.2 -4years and pediatric controls (n=2) of age range (0.2-0.3 years) were used for the miRNA and mRNA microarray study. The details of clinical samples including age, gender, laterality, tumor viability, clinical and histopathology details are mentioned in Table 1.

 

RNA isolation:

Total RNA isolation from retinoblastoma tumors and control pediatric retina samples were performed using Agilent Absolutely RNA miRNA kit (cat# 400814) according to manufacturer’s instructions. Total RNA was extracted from retinoblastoma cell lines (WERI-Rb1) using Trizol reagent (Invitrogen, Carlsbad, CA) and purified through RNA binding columns available with Agilent Absolutely RNA- miRNA kit (cat# 400814). We performed on- column DNase treatment at 37°C for 15 minutes, prior to elution. The total RNA purity was measured using Nano drop (ND-1000 UV-VIS Spectrophotometer) and the total RNA integrity for microarray was assessed using Agilent 2200 Tape Station (cat# G2964AA). The RNA quality was maintained consistent across samples with a RIN value > 6 for gene expression studies.

Tumor mRNA profiling:

Total RNA was isolated from 9 Rb tumors and 2 control pediatric retina samples using Agilent Absolutely RNA miRNA kit (cat# 400814) according to manufacturer’s instructions. Twenty-five nanograms of RNA from Rb tumors and control peadiatric retina samples were labelled with Cy3 dye using an Agilent Low Input Quick Amp Labelling Kit (p/n 5190-2305). Gene expression microarray analysis was performed using the Agilent SurePrint G3 Human GE 8 × 60K V2 Microarray and an Agilent SureScan Microarray scanner. The gene expression data were extracted using Agilent Feature Extraction Software (11.5.1.1) and analyzed using Agilent GeneSpring GX 13.1. The analysis was carried out using a t-test unpaired statistical method with Benjamini Hochberg FDR method.

Cell lines:

WERI-Rb1 & Y79 retinoblastoma cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in RPMI 1640 medium (Gibco, Cat #11875093), supplemented with 10% FBS and 1% Pen Strep (Penicillin –Streptomycin) and maintained at 37ºC in a humidified atmosphere of 5% CO_2, with intermittent shaking in an upright T25 flask.

Gene expression analysis:

Total RNA extracted for microarray from clinical subjects, were also used for RT PCR validation. RT-PCR was performed with Agilent Brilliant III Ultra-Fast RT-PCR reagent (cat# 600884), using Agilent AriaMX real time PCR instruments. Relative mRNA expression levels were quantified using the DDC(t) method (34). For in-vitro assays, total RNA was isolated from cells using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. 1µg of RNA was reverse transcribed using Bio-Rad iScript cDNA synthesis kit (cat# 1708890) and quantitative real time PCR was performed using Kappa Sybr fast qPCR kit (cat# KK4601) using Bio-rad CFX96 system. Relative mRNA expression levels were quantified using the DDC(t) method. Results were normalized to housekeeping human β-actin.

Results:

Advanced Rb tumors have unique EMT signatures.

We identified distinct dysregulated gene clusters, implicating gross differences between advanced intraocular tumour defined as AJCC Stage[17] cT3 or IIRC[18] Group E tumour (6089 genes, P<0.05, FC>2) and non-advanced intraocular tumour defined as AJCC Stage cT2 or IIRC Group D (7695 genes, P<0.05, FC>2) compared to pediatric retina. Notably, EMT transcription factors like ZEB1, SNAI2 and Twist were significantly upregulated in advanced tumors. High expression of these EMT transcription factors are associated with tumorigenic progression in other cancer systems[19, 20]. In addition to these findings, advanced Rb tumors demonstrated cadherin switching phenotype with high expression of N-cadherin and low expression of E-cadherin in tumor tissues, suggestive of EMT dissemination[21]. We speculate that advanced tumors maintain high expression of EMT to promote invasion and metastasis.

Rb cells confer high EMT program and metastasis

To extend our study of the consequences of RB1 downregulation in Rb tumors and its influence on EMT signatures, we overexpressed RB1 in Rb null Y79 and WERI-Rb1 cells. In real time gene expression assays, we found that Rb over expression decreased key EMT factors like ZEB1, Slug and N-cadherin in the immunoblot. However, Rb over expression increased E-cadherin expression indicating a halt in the EMT switch. We further confirmed the findings using RT-PCR that revealed a mesenchymal transition trend in control Rb null cells compared to Rb over expression. Our findings strongly suggest EMT as a modulator of mesenchymal phenotype to further promote invasion and migration.

Discussion

The present study identifies RB1 as a previously unrecognized regulator of EMT transcription factors in Rb tumors. We provide proof-of-concept evidence that mesenchymal transition in advanced Rb is likely to be halted by ectopic expression of RB1. Our focus here has been mainly on intraocular advanced and non-advanced retinoblastoma tumors, but our findings can be extended to other cancer systems that have persistent EMT mediated metastatic dissemination.

We identified the balance between EMT driven metastasis in Rb tumors to be influenced by RB1 gene. Recent reports support our findings, further highlighting the importance of RB1/ EMT circuits in cancers[22].  Advanced Rb tumors have high ZEB1 and Twist expression and these EMT TFs are master regulators of transition program and their oncogenic functions are widely studied in metastatic breast cancer[19], colon cancer[23] and glioblastoma[24]. During the EMT process, ZEB1 transcriptionally suppress the expression of its target genes, such as epithelial markers (E-cadherin) and correspondingly increasing the mesenchymal levels of vimentin and N-cadherin[25]. Transcriptionally, loss of E-cadherin is associated with upregulation of genes involved in mesenchymal transition like N-cadherin, Vimentin and transforming growth factor-β (TGFβ)[26]. Likewise, oncogenic activation of Twist along with SNAI1 can prevent cells from oncogene induced failsafe programs through RAS pathway[27]. We propose that key EMT drivers like ZEB1 and Twist acquires transcript stability due to the loss of function of RB1 gene in retinoblastoma.

As the Rb pathway is not druggable, an alternative approach is to mimic its function to understand RB1 regulated EMT circuits in Rb deficient cancers. This concept is further supported by our observations with ectopic expression RB1 in Y79 and WERI-Rb1 cells, that repress ZEB1 and Twist expression and its targets E-cadherin, N-cadherin and vimentin signaling cascade, inhibiting EMT transition and metastasis. In conclusion, our work reveals the mechanistic link between RB1 and EMT in advanced Rb tumors. Our study identifies RB1 as a potential target for EMT program and offers encouraging prospects for managing advanced retinoblastoma.

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