Abstract
Background: Patients with inflammatory bowel disease have markedly increased risk for developing colitis-associated colorectal adenocarcinoma (CAC). There is no established prognostic biomarker for CAC. Materials and Methods: A retrospective study was performed on a cohort of 57 CACs. Expression of caudal type homeobox transcription factor 2 (CDX2) and YES-associated protein 1 (YAP1) expression was correlated with clinicodemographic and histopathological features. Results: Neither YAP1 nor CDX2 expression alone was significantly associated with tumor invasion beyond the muscularis propria or lymph node status. However, a subgroup of CAC with double negativity for expression of YAP1 and CDX2 was more frequently found in younger patients, and more frequently associated with higher pathological tumor stage and nodal metastasis. Furthermore, a positive correlation between CDX2 and YAP1 expression was identified in CAC and sporadic colorectal adenocarcinoma. Conclusion: Our study demonstrates that double negativity for expression of YAP1 and CDX2 defines a subgroup of CAC with early onset and aggressive clinical features.
Patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, have lifelong chronic inflammation of the intestine, and therefore carry markedly increased risk for developing colitis-associated colorectal adenocarcinoma (CAC) (1-4). The underlying mechanism for the increased cancer susceptibility in patients with IBD remains unclear but the complicated crosstalk between the inflammatory environment, microbiome, and genetic factors likely contributes to this process. CAC accounts for 1-2% of all cases of colorectal adenocarcinoma and 15% of all deaths in patients with IBD (5). Currently, there is no established biomarker to predict the progression from IBD to CAC or prognosticate CAC, and the treatment options are limited.
The Hippo signaling pathway is an evolutionarily conserved tumor-suppressor pathway. Originally identified in Drosophila for its essential role in tissue size control, the Hippo signaling pathway regulates tissue homeostasis by coordinating cell proliferation, apoptosis and cell differentiation (6, 7). The expression of the Hippo signaling effector YES-associated protein 1 (YAP1) is dysregulated in multiple human malignancies, including colorectal adenocarcinoma. High expression of YAP1 is associated with tumor progression, drug resistance, and reduced survival in sporadic colorectal adenocarcinoma (8-11). Caudal type homeobox transcription factor 2 (CDX2), one of the master genes for intestinal epithelial development and differentiation, is a highly sensitive and specific marker for adenocarcinoma of intestinal origin (12). Down-regulation of CDX2 expression is observed in colorectal adenocarcinoma with poor differentiation or deficient DNA mismatch repair (MMR) (13, 14). Recent studies have identified CDX2 as a prognostic biomarker for sporadic colorectal adenocarcinoma (15-17). In this study, we aimed to investigate the expression and prognostic role of YAP1 and CDX2 in CAC.
Materials and Methods
Patient characteristics. The study was approved by the Institutional Review Board at the Cleveland Clinic Foundation (no. 05-100) in accord with the ethical standards established by the institution in which the experiments were performed in accord with the Helsinki Declaration of 1975. A retrospective study was performed on a cohort of 57 patients with CAC diagnosed between 1992 to 2010. All patients had a documented history of IBD. In addition, chronic colitis was confirmed by histopathological re-evaluation in all cases. Patients with sporadic colorectal adenocarcinoma and CAC with pre-operative neoadjuvant therapy were excluded from the study.
Clinicopathological characterization. Clinicopathological data were collected through reviewing medical records and tissue slides. Material included surgical resection specimens and biopsy specimens. Pathology reports and histology slides were examined to retrieve tumor information including tumor site, focality, differentiation, lymph node status, and tumor stage. All tumors were subjected to histological review using previously published criteria (18). Specific morphological features examined in this study include mucinous feature (presence of dysplastic mucin-producing epithelium with or without extravasated mucin), signet-ring feature (presence of prominent intracytoplasmic mucin vacuoles), medullary feature (presence of solid growth pattern, pushing border and prominent intraepithelial lymphocytes), Crohn-like feature (presence of discrete lymphoid aggregates), tumor heterogeneity (presence of different tumor clonal population with distinct morphological and phenotypic profiles), and dirty necrosis (presence of necrotic debris within the tumor glandular lumina).
Immunohistochemical analysis. Three tissue microarrays were constructed from representative areas of these CACs and subjected to immunohistochemistry. Antibodies used were against: YAP1 (Clone 63.7, diluted 1:300; Santa Cruz Biotechnology, Dallas, TX, USA), cytokeratin 7 (CK7) (Clone OV-TL 12/30, diluted 1:40; Dako, Carpinteria, CA, USA), CDX2 (Clone CDX2-88, diluted 1:10; BioGenex, Fremont, CA, USA), α-methylacyl-CoA racemase (AMACR) (Clone P504S, diluted 1:100; Zeta Corp, Arcadia, CA, USA), and β-catenin (Clone 14, diluted 1:250; BD Biosciences, San Jose, CA, USA). Positive and negative controls were included for the study. For CK7, cytoplasmic staining in ≥30% of tumor cells was considered positive. For YAP1, CDX2 and β-catenin, tumor cells with any nuclear immunoreactivity were considered positive. For AMACR, tumor cells with any cytoplasmic immunoreactivity were considered positive.
The Cancer Genome Atlas (TCGA) database. We analyzed the gene expression data from TCGA Colon Adenocarcinoma (TCGA-COAD) data collection (https://portal.gdc.cancer.gov). The correlation of CDX2 and YAP1 gene expression was analyzed by Pearson's correlation coefficient. Data were generated at GEPIA2 online (http://gepia2.cancer-pku.cn).
Quantitative real-time polymerase chain reaction (Q-PCR). In order to determine the effect of YAP1 on expression of CDX2, as a direct transcriptional target, the following cell lines were used in this study (19): Parental colorectal cancer cell line SW480, YAP1 knockout cell line SW480-YAP1-KO and YAP1 forced expression SW480 cell line YAP1-S127A. Total cellular RNA was extracted using the Direct-zol RNA Miniprep Plus Kit following the manufacturer's instructions (Zymo Research, Irvine, CA, USA). The purity and concentration of the RNA were evaluated by ultraviolet NanoDrop spectroscopy (Thermo Scientific, Waltham, MA, USA). RNA reverse transcription and real-time quantitative PCR (Q-PCR) were performed as described elsewhere (20). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene served as a housekeeping control gene. Data are expressed as the mean±SEM from four biological repeats. Primer sequences were: Human CDX2, forward: 5’-CGGCAGCCAAGTGAAAACCA-3’, reverse: 5’-GCGGTTCTGAAACCAGATTTTAACC-3’; human GAPDH forward: 5’-GATGACATCAAGAAGGTGGTGAAG-3’, reverse: 5’-TCCTTGGAGGCCATGTGGGCCAT-3’.
Clinico-demographics and histological features for 57 patients with colitis-associated colorectal adenocarcinoma.
Clinicopathological features of colitis-associated colorectal adenocarcinoma according to expression of YAP1 and CDX2.
Statistical analysis. Continuous variables were compared with Student's t-test. Categorical variables were summarized as count and percentage and compared with Fisher's exact test. A p-value of less than 0.05 was considered statistically significant.
Results
Patient characteristics. The study included 57 patients with CAC. The mean age of the patients was 51.1±15.7 years (range=17-83 years). Twenty-five patients were female. Of all patients, 24 (42.1%) had tumor invasion beyond the muscularis propria (pT3 or pT4), 24 (42.1%) had lymph node involvement (pN1 or pN2), 10 (17.5%) had multifocal lesions, and 22 (38.6%) had right-sided colon cancer. Among these CACs, 63.2% showed absence of dirty necrosis, 54.4% had mucinous feature, 14.0% had signet-ring feature, and 5.3% had medullary feature (Table I).
Expression and prognostic value of YAP1 and CDX2. YAP1 was expressed in 55.5% of CACs, and CDX2 was expressed in 70.2% of CACs. YAP1 expression in tumor was significantly associated with older age (p=0.029) and CDX2 positivity (p=0.001). On the other hand, tumors with no expression of YAP1 showed a weak trend of having signet-ring feature (p=0.144) and medullary feature (p=0.112). CDX2 expression was significantly associated with lack of dirty necrosis (p=0.036) and YAP1 positivity (p=0.001). The majority of CACs with YAP1 expression showed CDX2 expression (89.7%). In contrast, only half of the CACs negative for YAP1 expression showed CDX2 expression (50.0%). Neither YAP1 nor CDX2 expression alone was significantly associated with tumor invasion beyond the muscularis propria (pT3 or pT4) or lymph node status (Table II).
We further subcategorized the cohort based on the expression of YAP1 and CDX2. Notably, the subgroup of CACs that were negative for expression of YAP1 and CDX2, comprising 24.6% of the entire cohort, was more often found in younger patients with mean age of 45.3 years (p=0.036). Morphologically, this subgroup more frequently had dirty necrosis (p=0.049). Most importantly, this subgroup of CACs more frequently presented higher pathological tumor stage (92.9% with pT3/pT4), as compared with the rest of the cohort (62.8%; p=0.036). It was also more likely to have nodal metastasis (64.3% vs. 30.2% for the rest of the cohort, p=0.031) (Table III). Expression of YAP1 or CDX2 was not associated with other immunohistochemical markers such as CK7, AMACR, or nuclear beta-catenin immunoreactivity (Table II).
CDX2 as a transcriptional target of YAP1. Consistent with a positive correlation in CAC, analysis of TCGA database confirmed a positive correlation between YAP1 and CDX2 expression in sporadic colorectal adenocarcinoma (Figure 1A). YAP1 is a known transcriptional coactivator. To test whether YAP1 regulates CDX2 gene expression, Q-PCR was performed on the colorectal cancer cell line SW480. As shown in Figure 1B, CDX2 gene expression was induced by YAP1 overexpression, and was dramatically suppressed upon CRISPR-Cas9-mediated knockout of YAP1 gene, supporting the concept that CDX2 is a direct transcriptional target of YAP1.
Clinicopathological features of a colitis-associated colorectal adenocarcinoma subgroup double-negative for expression of YAP1 and CDX2.
Discussion
Patients with longstanding IBD have increased risk for developing CAC as compared to the general population, presumably through an ‘inflammation–dysplasia–carcinoma' sequence. Multiple lines of evidence suggest that this process is different from the well-studied adenoma–carcinoma pathway in sporadic colorectal adenocarcinoma (1-4). The risk factors for developing CAC includes the duration of IBD, the disease extent (pancolitis or left-sided colitis), the presence of pseudopolyposis, coexistence of primary sclerosing cholangitis, and family history of colorectal adenocarcinoma (21). However, the underlying mechanism for increased cancer susceptibility in patients with IBD remains unclear.
CAC has unique clinical features. It is more commonly seen in younger patients, frequently presents with multifocal lesions, left-side predominance, mucinous or signet-ring feature, and is associated with poor prognosis (1). Furthermore, the immunoprofile of CAC is also quite different from sporadic cases, with more frequent loss of CDX2 and special AT-rich sequence-binding protein 2 (SATB2) protein expression in CAC (22, 23). Loss of SATB2 is associated with poor prognosis in both sporadic adenocarcinoma and CAC (23-25). CDX2 is a homeobox gene that is expressed throughout the intestinal epithelium. As an intestine-specific transcription factor, CDX2 plays a pivotal role in regulating intestinal differentiation, and is widely used for the diagnosis of primary and metastatic colorectal adenocarcinoma (26, 27). CDX2 has also been identified as an independent prognostic biomarker in sporadic colorectal adenocarcinoma (15-17) but whether it plays similar role in CAC has not been examined.
Caudal type homeobox transcription factor 2 (CDX2) is a transcriptional target of YES-associated protein 1 (YAP1). A: YAP1 and CDX2 expression are positively correlated in colorectal adenocarcinoma. Data were generated at GEPIA2 online (http://gepia2.cancer-pku.cn) using The Cancer Genome Atlas (TCGA) datasets. TPM: Tags per million. B: YAP1 positively regulates CDX2 expression in colorectal cancer cell line SW480. Quantitative reverse transcription polymerase chain reaction was performed to measure CDX2 gene expression. Knockout of YAP1 (YAP1 KO) inhibited CDX2 expression in colorectal cancer cells, and constitutive overexpression of YAP1 (S127A) induced CDX2 expression in colorectal cancer cells. *Significantly different at p<0.05.
In the current study, we conducted a systemic clinicopathologicaI evaluation of CAC with two potential biomarkers, CDX2 and YAP1. Consistent with previous reports (22, 23), our study observed the loss of CDX2 expression in a specific population of CACs. However, unlike in sporadic colorectal adenocarcinoma (15-17), our study demonstrated that CDX2 alone is not significantly associated with tumor prognosis in CAC.
The Hippo signaling effector YAP1 is a potent transcriptional coactivator and activates target gene transcription through direct interaction with transcription factor TEA domain family members (TAED). YAP1 has been recognized as an important oncogene, and high level of YAP1 expression is associated with drug resistance and poor outcome in sporadic colorectal adenocarcinoma (8-11). Interestingly, our study demonstrates the presence of a CAC subgroup that is double negative for expression of YAP1 and CDX2. This subgroup comprised approximately one-fourth of the CAC patient population, and was associated with early onset and aggressive tumor behavior as indicated by higher tumor stage and frequent lymph node metastasis. Our study also provides evidence supporting the role of YAP1 as a transcriptional regulator for CDX2 gene expression. In contrast to the oncogenic function of YAP1 in sporadic colorectal adenocarcinoma (8-11), our study suggests a tumor-suppressive role of the YAP1 in CAC. Several studies have shown that YAP1 can serve as a tumor suppressor in human malignancies including colorectal adenocarcinoma (28), breast cancer (29), head and neck cancer (30), and hematological malignancy (31). In fact, YAP1 was shown to induce apoptosis through its interaction with p73 tumor suppressor in response to DNA damage (32-34). Whether the same molecular mechanism also mediates CAC tumorigenesis is unclear but YAP1-mediated gene transcription of the tumor suppressor CDX2 leads to a working hypothesis that in the context of chronic inflammation, the YAP1–CDX2 axis may have an important role in suppressing tumorigenesis in CAC.
In summary, our study reveals that the signaling pathway involving the YAP1–CDX2 axis may play a pivotal tumor-suppressive role in CAC, and the high-risk features of double-negative expression for YAP1/CDX2 suggests the need for routine screening of this subgroup in patients with CAC who may benefit from a more tailored neoadjuvant therapy and eventually a pathway-targeted therapy.
Footnotes
Authors' Contributions
FY, JL and XL planed the study. FY and HX conducted the statistical analysis. YC and JD performed experiments. FY drafted the article. HX, JL, JD, and XZ critically reviewed the draft. All Authors approved the final version of the article.
Conflicts of Interest
The Authors declare no competing interests in this study.
- Received June 18, 2020.
- Revision received July 9, 2020.
- Accepted July 13, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
