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NAP1L1: a Novel Human Colorectal Cancer Biomarker Derived from Animal Models of Apc Inactivation

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NAP1L1: a Novel Human Colorectal Cancer Biomarker Derived from Animal Models of Apc Inactivation fonc-10-01565 August 7, 2020 Time: 19:2 # 1 ORIGINAL RESEARCH published: 11 August 2020 doi: 10.3389/fonc.2020.01565 NAP1L1: A Novel Human Colorectal Cancer Biomarker Derived From Animal Models of Apc Inactivation Cleberson J. S. Queiroz1,2, Fei Song1,3, Karen R. Reed4,5, Nadeem Al-Khafaji1, Alan R. Clarke5†, Dale Vimalachandran6, Fabio Miyajima1,7, D. Mark Pritchard1* and John R. Jenkins1 1 Institute of Systems, Molecular and Integrative Biology, Henry Wellcome Laboratory, University of Liverpool, Liverpool, United Kingdom, 2 Faculty of Medicine, Federal University of Mato Grosso (UFMT), Cuiaba, Brazil, 3 INFRAFRONTIER GmbH, Neuherberg, Germany, 4 Wales Gene Park, Division of Cancer and Genetics, Cardiff University School of Medicine, Cardiff, United Kingdom, 5 European Cancer Stem Cell Research Institute, Cardiff University School of Biosciences, Cardiff, United Kingdom, 6 Department of Colorectal Surgery, Countess of Chester Hospital NHS Foundation Trust, Chester, Edited by: United Kingdom, 7 Molecular Epidemiology Laboratory, Oswaldo Cruz Foundation, Eusebio, Brazil Boris Zhivotovsky, Karolinska Institutet (KI), Sweden Introduction: Colorectal cancer (CRC) is the second leading cause of cancer death Reviewed by: Giovanna Caderni, worldwide and most deaths result from metastases. We have analyzed animal models University of Florence, Italy in which Apc, a gene that is frequently mutated during the early stages of colorectal Gabriele Multhoff, carcinogenesis, was inactivated and human samples to try to identify novel potential Technical University of Munich, Germany biomarkers for CRC. *Correspondence: Materials and Methods: We initially compared the proteomic and transcriptomic D. Mark Pritchard [email protected]; profiles of the small intestinal epithelium of transgenic mice in which Apc and/or Myc [email protected] had been inactivated. We then studied the mRNA and immunohistochemical expression †Deceased of one protein that we identified to show altered expression following Apc inactivation, nucleosome assembly protein 1–like 1 (NAP1L1) in human CRC samples and performed Specialty section: This article was submitted to a prognostic correlation between biomarker expression and survival in CRC patients. Molecular and Cellular Oncology, a section of the journal Results: Nap1l1 mRNA expression was increased in mouse small intestine following Frontiers in Oncology Apc deletion in a Myc dependant manner and was also increased in human CRC Received: 11 December 2019 samples. Immunohistochemical NAP1L1 expression was decreased in human CRC Accepted: 20 July 2020 Published: 11 August 2020 samples relative to matched adjacent normal colonic tissue. In a separate cohort of Citation: 75 CRC patients, we found a strong correlation between NAP1L1 nuclear expression Queiroz CJS, Song F, Reed KR, and overall survival in those patients who had stage III and IV cancers. Al-Khafaji N, Clarke AR, Vimalachandran D, Miyajima F, Conclusion: NAP1L1 expression is increased in the mouse small intestine following Pritchard DM and Jenkins JR (2020) Apc inactivation and its expression is also altered in human CRC. Immunohistochemical NAP1L1: A Novel Human Colorectal Cancer Biomarker Derived From NAP1L1 nuclear expression correlated with overall survival in a cohort of CRC patients. Animal Models of Apc Inactivation. Further studies are now required to clarify the role of this protein in CRC. Front. Oncol. 10:1565. doi: 10.3389/fonc.2020.01565 Keywords: colorectal cancer, biomarkers, Apc, NAP1L1, prognosis, survival Frontiers in Oncology| www.frontiersin.org 1 August 2020| Volume 10| Article 1565 fonc-10-01565 August 7, 2020 Time: 19:2 # 2 Queiroz et al. NAP1L1: A Novel CRC Biomarker INTRODUCTION to identify Myc dependant Wnt pathway genes following Apc inactivation. We investigated whether there were any Colorectal cancer (CRC) is the third most common cancer type genes/proteins that showed congruent findings in both and the second leading cause of cancer death worldwide (1). analyses according to strict criteria. One protein, nucleosome Deaths usually result from recurrent and metastatic disease. Most assembly protein 1 like 1 (NAP1L1) was identified that international guidelines recommend chemotherapy to reduce met our criteria. the risk of recurrence in stage III tumors and to prolong We therefore analyzed the expression of Nap1l1 in Apc– survival in stage IV cancers (2). Conversely, chemotherapy is deficient (AhCreCApcfl=flMycC=C) and double-mutant Apc-Myc generally not used in stage I and most stage II tumors. However, deficient (AhCreCApcfl=flMycfl=fl) mice to assess whether its some patients with low-risk stage III disease may respond well expression was Myc-dependant and therefore a potential following courses of chemotherapy that are shorter than the biomarker of Wnt activation. Following confirmation of our 6-month standard schedule, although the definition of “low- findings, we investigated the mRNA and protein expression risk” has not been well established in this scenario (3,4). of NAP1L1 in tumor and adjacent normal mucosa samples Additionally, almost 20% of patients who have stage II tumors from patients with CRC as well as colonic tissues from and who are therefore considered to have low-risk disease, unaffected individuals. relapse and eventually die from cancer progression (5). There is currently no accurate biomarker to better define prognosis within stage groups. Therefore, biomarkers for prognostic MATERIALS AND METHODS stratification in CRC have the potential of improving the treatment decision-making process (4,6). We hypothesized that Animal Samples studying molecular mechanisms that are known to be involved Mouse experiments were performed with UK Home Office in CRC development might yield promising novel biomarkers approval with personal and project licenses (30/2737 and for this disease. 30/3279) and according to ARRIVE guidelines and following Adenomatous polyposis coli (APC) inactivating mutations local ethical review by the Cardiff University Animal Welfare are the earliest and most common genetic alterations in the Ethical Review Panel. The transgenic mice that were used in this adenoma-carcinoma sequence that leads to CRC (7). Such study were generated and maintained at the University of Cardiff mutations result in the accumulation of b-catenin within cells as previously described in (14). Small intestinal epithelial cell and activation of the Wnt signaling pathway (8). Animal extracts were generated from these mice following injection of models of Apc inactivation demonstrate epithelial transformation beta-naphthlaflavone as described by Hammoudi et al. (16). and tumor formation mirroring cancer development (9– 11). One of these models is the AhCreCApcfl=fl mouse, qPCR RT-PCR an animal bearing loxp-flanked Apc alleles and a Cre- For the mouse small intestinal tissue samples and human recombinase transgene. When this animal is exposed to CRC samples obtained in the United Kingdom (Wales cohorts b-naphthoflavone, Cre-recombinase transcription is activated 1 and 2), total RNA was used to synthesize first strand resulting in the deletion of loxp-flanked Apc alleles specifically cDNA using a VersoTM cDNA Kit (Thermo Scientific) and in the small intestinal epithelium, thus causing an acute anchored oligo-dT primers (Thermo Scientific) according to activation of the Wnt pathway (9). The ApcMin=C mouse has the manufacturer’s instructions. Single-stranded cDNA samples a germline mutation in one Apc allele simulating a familial were amplified in a Polymerase chain reaction (PCR) using adenomatous polyposis (FAP) patient, and spontaneously sequence-specific primers (Eurogentec) and probes from the develops multiple intestinal neoplasms during its life-span (12, Universal Probe Library (Roche) that were designed using the 13). The Myc gene is a Wnt-target that plays an essential Universal ProbeLibrary Assay Design Centre, using PCR Master role in the development of malignant phenotypes upon Apc mix (Roche) and a light cycler 480 (Roche) (see Supplementary inactivation (14, 15). We hypothesized that the analysis of Table 2 for primers and probes used). mouse models of Apc and Apc/Myc deletion could lead to the For the human CRC samples recruited in Brazil, RNA discovery of genes or proteins with potential clinical use as was extracted using the RNeasy R Mini Kit (Qiagen, Hilden, human CRC biomarkers. Germany). cDNA was produced using TaqMan R Reverse Our group has previously published a proteomic evaluation Transcription Reagents kit (Applied Biosystems, Carlsbad, of an animal model of CRC based on acute Apc deletion CA, United States) according to the manufacturer’s protocol. (AhCreCApcfl=fl mouse) (16). A 4-plex iTRAQ labeling analysis Quantitative PCR reactions were performed using the 7500 Fast identified 126 proteins that were significantly altered upon Real-Time PCR System (Applied Biosystems, Foster City, CA, Apc deletion (76 up- and 50 down-regulated) and which United States) (see Supplementary Table 3 for expression assay were proposed as Wnt targets. We have now performed an specifications). additional analysis of this dataset by comparing the protein list with the transcriptomic data generated using Affymetrix Proteomic and Microarray Comparison arrays and intestinal tissues from the same mice (14). This Analysis study used Apc–deficient (AhCreCApcfl=flMycC=C) and double- We set out to determine the subset of genes/proteins, mutant Apc-Myc deficient (AhCreCApcfl=flMycfl=fl) mice which demonstrated upregulation of both protein and mRNA Frontiers
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