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Original Article Prognostic Impact of Cancer Stem Cell Markers ABCB1, NEO1 and HIST1H2AE in Colorectal Cancer

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Original Article Prognostic Impact of Cancer Stem Cell Markers ABCB1, NEO1 and HIST1H2AE in Colorectal Cancer Am J Transl Res 2020;12(9):5797-5807 www.ajtr.org /ISSN:1943-8141/AJTR0100791 Original Article Prognostic impact of cancer stem cell markers ABCB1, NEO1 and HIST1H2AE in colorectal cancer Bogdan Badic1, Stéphanie Durand2*, Flaria El Khoury2&, Pierre De La Grange4, David Gentien3, Brigitte Simon2, Catherine Le Jossic-Corcos2#, Laurent Corcos2# 1INSERM UMR 1101, 2INSERM UMR 1078, Université de Brest, 22 Avenue Camille Desmoulins, 29238 Brest, France; 3Institut Curie, Département de Recherche Translationnelle, Plateforme Génomique, 1 Avenue Claude Vellefaux, 75010 Paris, France; 4GenoSplice Technology, 19 Rue Claude Bernard, 75005 Paris, France; &Present address: CNRS UMR 9197, Neuro-PSI, 1 Avenue de la Terrasse, 91198 Gif-Sur-Yvette, France; *Present ad- dress: EA7500, Université de Limoges, Faculté des Sciences et Techniques, 123 Avenue Albert Thomas, 87060 Limoges, France. #Equal contributors. Received August 10, 2019; Accepted March 23, 2020; Epub September 15, 2020; Published September 30, 2020 Abstract: Colon cancer develops according to a defined temporal sequence of genetic and epigenetic molecular events that may primarily affect cancer stem cells. In an attempt to identify new markers of such cells that would help predict patient outcome, we performed a comparative transcriptome analysis of colon cancer stem cells and normal colon stem cells. We identified 162 mRNAs, either over- or under-expressed. According to Cox multivariate regression with our set of 83 colorectal cancers, low expression of ABCB1, NEO1, tumor size and the presence of distant metastases were predictive factors for overall survival. Combined expression of ABCC1 and NEO1 was a sig- nificant predictor for overall survival in our cohort, which was confirmed by external validation in 221 colorectal can- cers from the Cancer Genome Atlas (TCGA) portal. Tumor size, lymph node involvement and HIST1H2AE expression were also independently correlated with disease-free survival. Taken together, our results suggest that molecular markers of colorectal cancers ABCB1, NEO1 and HIST1H2AE are prognostic factors in colorectal cancer patients. It can be proposed that surveying expression of these marker genes should help better characterizing CRC prognosis, and help selecting the best therapeutic options. Keywords: Colorectal cancer, cancer stem cell, ABCB1, NEO1, HIST1H2AE, prognostic factors, whole transcrip- tome analysis Introduction Chemotherapy is the standard treatment for metastatic colorectal cancer as it prolongs Colorectal cancer (CRC) is the third most com- survival and improves life quality [6]. Whereas mon cancer in men and the second in women objective response rates were only 20 to 30% worldwide [1]. Incidence is low before the age with the combination of 5-fluoro-uracile/folinic of 50, but strongly increases thereafter. Me- acid, the addition of new drugs, such as oxali- dian age at diagnosis is about 70 in developed platin or irinotecan, helped increasing respon- countries [2]. CRC has a good prognosis when se rates up to around 50% and improved me- diagnosed at an early stage: the 5-year relative dian survival from 6 months to about 2 years survival is 91% for localized stages and 70% [7, 8]. The emergence of targeted therapies, in case of loco-regional invasion [3]. However, such as Epidermal Growth Factor (EGFR) rece- 5-year survival drops down to roughly 11% in ptor inhibitors (Cetuximab) or angiogenesis in- metastatic situations, which represent approxi- hibitors (Bevacizumab) improved these figures mately 25% of patients at diagnosis [4]. The further [9]. However, the 5-year survival rate death rate from CRC has decreased over the of patients treated with chemotherapy alone last 20 years, thanks to better disease man- remains below 1% [10]. In this context, it is agement (early diagnosis and improvement of imperative to better comprehend the develop- therapeutic modalities), but remains devastat- mental process of cancer lesions and to move ing worldwide [5]. towards more individualized treatments. Cancer stem cell molecular predictors of colorectal cancer It is believed that colon stem cells from the 2014.1 annotations [13]. Transcriptomic data bottom of the intestinal crypts can undergo were normalized using quantile normalization. molecular changes that make them turn into Background corrections were made with anti- a “cancer-founder” cell population resistant to genomic probes selected as described [14, conventional anticancer therapies [11]. Never- 15]. Only probes targeting exons annotated theless, the distinction between normal and from FAST DB® transcripts were selected to cancer stem cells is not well defined, and it is focus on well-annotated genes whose mRNA difficult to recognize cell markers specific to sequences were in public databases. Bad- the cancer-initiating population. According to quality selected probes (e.g. probes named by the cancer stem cell hypothesis for the origin Affymetrix as ‘cross-hybridizing’) and probes of aggressive CRCs, surveying such markers whose intensity signal was too low compared could be a tractable way to predict cancer out- to anti-genomic background probes with the come [12]. Although this could be achieved on same GC content were removed from the an- a per case basis, by analyzing the putative alysis. Only probes with a Detection Above links of candidate genes with colorectal can- BackGround (DABG) p-value ≤ 0.05 in at least cer, a blind, transcriptome-wide survey would half of the arrays were considered for statisti- address this question in a more comprehen- cal analysis. Only genes expressed in at least sive way. In the present study, we conducted a one compared condition were analyzed. Gene differential transcriptome analysis between expression was recorded only if at least half of stem cell populations derived from normal co- the gene probes had a DABG p-value ≤ 0.05. lons and from cancerous colons. This approach Unpaired Student’s t-tests were performed to led us to sort out several markers specific for compare gene intensities in the different bio- the cancer stem cell compartment. We ana- logical replicates. Genes were considered as lyzed expression of these markers in colorectal significantly regulated when fold-changes were tumors, together with that of already reported ≥ 1.5 and raw p-values ≤ 0.05. CRC markers, and confronted these data to histopathology characteristics and patient sur- Real-time PCR analyses vival. Using this approach, we identified novel molecular markers associated with either dis- To evaluate the prognostic impact of the tran- ease-free (DFS) or overall survival (OS). script content in CRC compared to healthy tis- sue, the differential expression of 21 selected Materials and methods genes was analyzed by real-time PCR (StepOne plus, Applied Biosystems). Briefly, total RNA Gene-expression profiling was extracted using NucleoSpin® RNA kit (Macherey-Nagel, Hoerdt, France) according to Stem cell populations from either disease-free the manufacturer’s instructions. Complemen- colon tissues or colon cancers were purcha- tary DNA synthesis and real-time PCR were per- TM sed from Celprogen (3 populations of each, formed as described [16]. All conditions were Torrance, CA) and grown according to the man- normalized relative to the RPLP0 (ribosomal ufacturer’s instructions. Stem cells were from protein P0) control RNA. PCR primers were pur- male donors of Caucasian origin with a mean chased from Eurogentec (Seraing, Belgium). age of 60. Total RNA was extracted with TRI- Primer sequences will be made available on zol® reagent (ThermoFisher scientific, Illkirch, request. The results were analyzed using the France) according to the manufacturer’s ins- ΔΔCt method [17]. tructions and quality controls were performed using the RNA 6000 Nano LabChip® and the Patients and clinical data 2100 BioAnalyzer (Agilent Technologies, Mas- sy, France). GeneChip® Human Transcriptome All patients signed an informed consent form Array 2.0 (HTA2.0, Affymetrix, Santa Clara, CA, in which they granted use of data obtained USA) hybridization was performed at the Curie from analysis of their tissue samples. Eighty- Institute microarray core facility (Paris, France) three patients from our institution were in- according to Affymetrix procedures. Affymetrix cluded into the study: 36 women (43%) and 47 HTA2.0 dataset analysis and visualization were men (57%), with an average follow-up of 32.7 performed using EASANA® (GenoSplice tech- (1-117) months. The average age of the pati- nology), based on the GenoSplice’s FAST DB® ents was 71 (26-94) with an average American 5798 Am J Transl Res 2020;12(9):5797-5807 Cancer stem cell molecular predictors of colorectal cancer Society of Anesthesiologists physical status All clinical and gene expression data were score of 2.64. Tumors were localized in the used in a multivariate Cox regression analysis. right colon in 38 (46%) patients, in the left co- In order to evaluate the improvement in pro- lon in 29 (35%) patients, and in the rectum in gnosis stratification, models combining genes 16 (19%) patients. The preoperative extension expression with their optimal cut-off values assessment was positive with the discovery of were tested among the two cohorts. The re- synchronous liver lesions for 13 (16%) patients sulting Kaplan-Meier curves were compared and pulmonary lesions for 7 (8%) patients. using median OS in each group, hazard ratios (HR) and associated 95% confidence intervals External validation cohort (CI). Higher values of HR, with 1 being exclud-
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