Frequent Activation of Notch Signaling Pathway in Colorectal Cancers and Its Implication in Patient Survival Outcome

Colorectal cancer is a major health concern as it ranks third in incidence and second major cause of cancer-related deaths worldwide. A leading cause of treatment failure has been attributed to cancer stem cells that can invariably resist existing chemotherapeutic regimens. Notch signaling pathway has been involved in the maintenance of stem cells besides being crucial in cell fate decision and embryonic development. This pathway has also been implicated in several human malignancies including colorectal cancer. We investigated mRNA expression of four Notch receptors (Notch1–4), five ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), and four target genes (Hes1, Hes5, Hey1, and Hey2) using highly specific TaqMan gene expression assays in colorectal adenomas and cancers. Upregulated expression of Notch receptors ranged between 29 and 73% in colorectal cancers and between 11 and 56% in adenomas. Expression of Notch3 and Notch4 receptors was significantly higher in colorectal cancers compared to normal and adenoma tissues. The Jagged and Delta-like ligands were overexpressed between 25 and 52% in colorectal cancers, while in adenomas, it ranged between 0 and 33%. Combining the data for upregulation of receptors and ligands suggests that 86% colorectal cancers and 56% adenomas exhibited overexpression of Notch pathway genes in our cohort. Notch target genes were upregulated between 24 and 33% in colorectal cancers and between 11 and 22% in adenomas. Collating upregulation of Notch receptors and ligands with the target genes showed concordance in 58% colorectal tumors. Additionally, we evaluated expression of Notch receptors, ligands, and target genes with prognosis using the TCGA mRNA expression dataset. Patients overexpressing Notch3, Notch4, and Hey1 had significantly poorer overall survival relative to those having lower levels of these genes. Taken together, Notch signaling components are aberrantly overexpressed in colorectal tumors, and development of therapeutics targeting the Notch pathway may prove to be beneficial in the management of colorectal cancers.


Introduction
Colorectal cancer (CRC) is the second most common cancer in women and ranks third in men worldwide [1]. CRC is a group that includes cancers of the colon, rectum, and anus.
For both sexes combined, CRC is the third most commonly diagnosed cancer representing 10.2% of the total cases and second leading cause of cancer-related death (9.2%) worldwide in 2018 [1]. In Saudi Arabia, it is the most common cancer in males (19.6%), while in female, it ranks number three (9.5%). e cancer-related mortality in Saudi Arabia is highest for patients with CRC accounting for 15.2% of all deaths due to cancer [2].
Distant metastasis is the main cause of colorectal cancerrelated death [3]. Despite several advances in early diagnosis and treatments that include chemotherapy, immunotherapies, antiangiogenics, and surgical treatment, a significant percentage of early-stage cases will develop metastases and eventually die. is resistance to therapy eventually leading to fatality has been mainly attributed to cancer stem cells (CSCs). Understating the mechanism about how these cells are able to resist existing treatments may lead to the development of new therapies targeting CSCs [4].
Several molecular pathways are known to be involved in the functioning of CSCs including Wnt, Hedgehog, and Notch signaling [5]. Notch is a highly conserved signaling pathway that plays an important role in embryogenesis and cell fate determination whose genes were discovered in 1917 as a result of abnormal formation of notches in the wings of Drosophila melanogaster [6,7]. Besides implication in several developmental defects in humans, this pathway has been implicated in the pathogenesis of leukemia and several other cancers including colorectal cancers [8][9][10]. Aberrantly activated Notch signaling plays a crucial role in carcinogenesis and progression of various human malignancies including the breast [11] and colorectal cancer [12]. e overexpressions of Notch signaling components are correlated with CRC progression and metastasis [13]. However, the involvement of Notch signaling in early-stage CRC has not been well understood, and only few studies have been undertaken to investigate its role [14,15]. Interestingly, besides oncogenic role associated with tumor progression and metastasis, Notch also functions as tumor suppressor [16]. In mammals, the Notch pathway consists of five ligands and four receptors, which are single-pass transmembrane proteins. e five ligands are members of the Delta/Serrate/LAG-2 (DSL) family of proteins and include Delta-like-1 (Dll-1), Delta-like-3 (Dll-3), Delta-like-4 (Dll-4), Jagged-1, and Jagged-2, while receptors include Notch1, Notch2, Notch3, and Notch4 [17].
Activation of Notch pathway occurs via the binding of Notch ligands with their receptors. is activation leads to a proteolytic cleavage of the Notch protein via disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) and disintegrin and metalloproteinase domaincontaining protein 10 (ADAM10) in the extracellular domain followed by the c-secretase enzyme complex cleaving the transmembrane domain. As a result, Notch intracellular domain (NICD) is released and translocated to the nucleus forming a complex with transcription factor CSL and transcriptional coactivators including mastermind-like polypeptides (MAML) and recombination signal binding protein for immunoglobulin kappa J region (RBPj). is interaction leads to the activation of various target genes associated with cell survival, differentiation, cell growth, and tumorigenesis [18].
In this study, we investigated the involvement of the Notch signaling pathway in colorectal adenomas and cancers arising in Saudi Arabian patients by examining the expression of Notch receptors, ligands, and target genes. Additionally, using the mRNA expression data from TCGA, we studied the correlation of Notch pathway gene expression with the survival outcome of colorectal cancer patients.

Patient Samples.
Tissue biopsies from colorectal cancers (n � 66), adenomas (n � 9), and corresponding adjacent normal were obtained from the Gastroenterology division of King Khalid University Hospital (KKUH), Riyadh, Saudi Arabia. e specimens were collected and stored in RNA stabilizing reagent (Qiagen, Germany) at 4°C for 24 h and then at − 80°C till the time of RNA extraction. Samples were obtained from the patients prior to receiving chemotherapy or radiation therapy. e Ethics Committee of KKUH approved the study, and written informed consent from the patients was obtained.

RNA Extraction and cDNA Synthesis.
Tissues were homogenized in a Dispomix tube by Dispomix drive (Medic Tools, AG, Zug, Switzerland). AllPrep DNA/RNA Mini Kit (Qiagen, Germany) was used to extract total RNA as per the manufacturer. On-column DNaseI treatment was performed using RNase-Free DNase Set (Qiagen, Germany). RNA integrity was determined by RNA 6000 Nano kit and 2100 Bioanalyzer system (Agilent Technologies Inc., California, USA). RNA quantitation was performed on NanoDrop ™ 8000 Spectrophotometer ( ermo Fisher Scientific). RNAs were reverse-transcribed to obtain cDNA using high-capacity cDNA reverse transcription kit ( ermo Fisher Scientific) according to the manufacturer and stored at − 20°C.

Real-Time RT-PCR.
Real-time quantitative PCR reactions were performed in triplicate for each gene in a final volume of 10 μl. Each reaction contains 0.5 μl of 20X Taq-Man gene expression assay, 5.0 μl of 2X TaqMan gene expression master mix, 2.0 μl cDNA, and 2.5 μl nuclease-free water. Amplification was performed using QuantStudio ™ 7 Flex Real-Time PCR System (Applied Biosystems) at the following reaction conditions: AmpliTaq Gold DNA Polymerase hot start activation at 95°C for 30 s and 40 cycles at 95°C for 15 s and 60°C for 60 s. TaqMan assay IDs for the Notch pathway genes and GAPDH that was used as housekeeping gene are shown in Table 1. mRNA expression fold change in colorectal cancers and adenomas relative to adjacent normal tissues was calculated using the 2 − ΔΔCt method. Additionally, we also calculated expression of each gene by using the 2 ΔCt method using GAPDH as reference gene in normal, adenoma, and cancer tissues.

Kaplan-Meier Analysis.
To evaluate the effect of Notch pathway gene expression on the prognosis of colon adenocarcinoma patients, Kaplan-Meier survival analysis was performed using mRNA expression data from studies performed by e Cancer Genome Atlas (TCGA) obtained from OncoLnc, http://www.oncolnc.org. Patients were sorted according to gene expression levels, and bottom third (n � 145) versus top third (n � 145) expressers were compared; hence, lower and upper percentiles were set as 33 : 33 for the survival curve analysis.

Statistical Analysis.
Statistical significance for the expression of Notch pathway components between normal, adenoma, and colorectal cancer was determined using the two-tailed Student's t-test. e survival rate was calculated using Kaplan-Meier estimator and was assessed by the logrank test on OncoLnc (http://www.oncolnc.org/), an online analysis tool. A p value <0.05 was considered to indicate statistical significance.
Activation of Notch signaling can occur as a result of abnormal overexpression of either of the Notch receptors or ligands. Combining the data for the deregulated expression of these components suggests that the pathway activation could be detected in 86% (57/66) of CRCs and 56% (5/9) of adenomas in our cohort. e difference between activation of the Notch pathway in adenomas versus colorectal cancers was found to be statistically significant (p � 0.0431).
Besides calculating fold-change expression of Notch pathway genes in adenomas and colorectal cancers relative to the adjacent normal tissues, we further calculated expression of Notch3 and Notch4 by the ΔCt method using GAPDH as reference gene in normal, adenoma, and cancer tissues. Notch3 and Notch4 expressions were significantly higher in colorectal cancers compared to normal and adenoma tissues (Notch3: normal vs. CRC, p � 0.0052, adenoma vs CRC, p � 0.0018; Notch4; normal vs. CRC, p � 0.0046, adenoma vs. CRC, p � 0.036) (Figure 2). Furthermore, we compared Notch pathway gene expression based on clinical stages of the 43 colorectal tumors for which TNM staging information was available. None of the examined genes showed significant differences in their expression in early-stage (Stages I and II) compared to latestage (Stages III and IV) cancers ( Figure 3).

Association of Notch Pathway Gene Expression with
Prognosis.
e survival correlation of colon adenocarcinoma patients with Notch pathway gene mRNA expression was obtained from OncoLnc, a web-based tool publicly available at http://www.oncolnc.org that contains survival data of 21 cancers from studies performed by e Cancer Genome Atlas (TCGA). Of the 13 Notch pathway genes that we investigated, mRNA expression of Notch3, Notch4, and Hey1 was significantly associated with survival. Colon adenocarcinoma patients with a higher mRNA expression of Notch3, Notch4, and Hey1 presented significantly a shorter overall survival compared to those with low expression (Figure 4).

Discussion
Notch signaling pathway plays an important role in controlling the stability between cell proliferation, apoptosis, and differentiation. erefore, it has been proposed that this signaling might be responsible for human tumor development and progression as it has been commonly altered in several malignancies [19]. As a result, Notch signaling pathway proteins can be exploited for novel cancer therapeutic targets [20]. CRC has frequent genetic and physiological alterations that may affect proliferation and cell survival, and Notch signaling is known to be one of the common alterations. is signaling has the ability to control the homeostatic self-renewal of intestinal epithelium as well as its tumorigenic transformation as both these processes are extraordinarily symmetrical [21]. In the control of intestinal cell fate, for instance, the decision between secretary and adsorptive cells can be regulated via the expression of Hes1, one of the Notch target genes [21]. us, its deregulated expression may lead to abnormality in the intestine. Our data show that Hes1 is upregulated ≥2-fold in 33% of CRCs while was overexpressed in 22% in adenomas. Several investigations indicate that overexpression of Notch1, Jag1, and Jag2 was found in human intestinal adenomas, implying that elevated Notch signaling might be responsible for colorectal cancer initiation [22][23][24]. Apart from activating mutations of Notch1 in T-cell acute lymphoblastic leukemia (T-ALL), somatic mutations in Notch1, 2, 3, and 4 genes in common solid cancers are rare [8,25]. However, using datasets from the Cancer Cell Line Encyclopedia that contains mutational status of 1651 genes across 905 cancer cell lines and datasets on the cBioPortal for more than 2900 primary tumors, Mutvei et al. showed that the mutation frequency of Notch receptor and ligand genes are similar to that of established oncogenes but are higher in cell lines than in associated primary tumors [26]. Abnormal overexpression of Notch receptors or ligands leads to the activation of Notch signaling in CRC. For instance, our data showed that Notch1 was overexpressed ≥2fold in 36% of CRCs, while Jag2 was found to be the predominant ligand that was overexpressed in 52% compared with Jag1 upregulation in 26% of CRCs. Several studies have demonstrated antitumor efficacy by therapeutically targeting the Notch signaling pathway. Concurrently targeting Notch and Erbb2 pathways in metastatic colorectal cancers that were resistant to cetuximab resulted in antitumor response, suggesting dependence of tumor on Notch and Erbb2 [27]. Additionally, targeting Dll4-Notch signaling using anti-Dll4 either alone or in combination with irinotecan showed  efficacy in mutant KRAS colorectal xenograft tumors that were insensitive to cetuximab [28]. Anti-Dll4 antibody has also shown to inhibit breast cancer growth [29]. Furthermore, in KRAS-mutant lung adenocarcinoma, combined inhibition of DDR1 and Notch signaling resulted in regression of patient-derived xenografts with an efficacy comparable to standard chemotherapy [30]. Mullendore and coauthors observed overexpression of Notch ligands, predominantly Jag2 and Dll4 that were observed in 90% and 50% pancreatic cancer cell lines, respectively. ey suggested that sustained activation of Notch signaling is required for the maintenance of pancreatic cancer as inhibition of Notch signaling abrogated anchorage-independent growth of pancreatic cancer cells [31]. Gamma-secretase cleaved activated Notch1 and Notch3 proteins have been detected in majority of breast cancer cell lines. Downregulation of Notch3 but not Notch1 considerably suppressed proliferation and induced apoptosis of the Erbb2-negative breast cancer cell lines implicating Notch3-mediated signaling in the proliferation of these cells [32]. Bernasconi-Elias and colleagues generated Notch3 inhibitory antibodies that exhibited strong antileukemic activity in cell lines as well as tumor xenografts with Notch3 activating mutations [33]. Notch3 gene amplification resulting in significant protein overexpression has been linked with proliferation and survival of ovarian carcinomas as inactivation of Notch3 suppressed proliferation and promoted apoptosis in cell lines overexpressing Notch3 [34]. Rallis G et al. showed that the overexpression of Notch3 in CRC patients was associated with bad outcomes, with about 2.5 times reduced disease-   free survival (DFS) and overall survival (OS) [35]. Another study also confirmed a link between high levels of Notch3 and aggressive malignant colorectal cancer cell phenotype [36] and tumor recurrence after surgical resection of CRC [37,38]. Our data indicate that aberrant Notch3 overexpression was observed in high proportion of colorectal tumors seen in 73% cases, and its expression was drastically associated with poor survival (p � 0.0101). In view of these observations, inactivation of Notch3 signaling may prove beneficial in the therapeutic outcome of colorectal cancers. Guiyang Wu and colleagues point out that the upregulation of Notch4 was significant in cancer tissues and liver metastases compared with adjacent normal tissues. Also, they emphasize that Notch4 gene is abnormally overexpressed in CRC and is a prospective biomarker for survival of CRC patients [39]. We observed overexpression of Notch4 in 56% of colorectal cancers. In our cohort, Notch4 expression was significantly higher in colorectal cancers than normal (p � 0.0046) as well as adenoma tissues (p � 0.036), suggesting its involvement in CRC progression. Furthermore, in our analysis, with the TCGA dataset, Notch4 overexpression was found to be drastically associated with poor survival (p � 0.0275).
Han and coauthors demonstrated overexpression of Hey1 in CRC tissues and cell lines and attributed it to downregulation of miR-769 that directly targets Hey1 [40]. Furthermore, they found that the overexpression of this downstream effector of the Notch signaling pathway was significantly higher in advanced stage CRCs. Aberrant regulation of Hey1 levels through miR-769 leads to deregulation of several biological processes such as increased cell proliferation and invasion in colorectal cancers [40]. Our data reveal that Hey1 was overexpressed ≥2-fold in 24% of CRCs. Additionally, the Hey1 target gene of Notch signaling was significantly associated with poor survival (p � 0.0394). Overall, our data demonstrated that deregulation of Notch signaling was detected in 56% of adenomas and 86% of CRCs in our cohort. ough our sample size is small and results need to be interpreted cautiously, activation of the Notch pathway in colorectal cancers versus adenomas is significantly different (p � 0.0431), implying it to be activated in the progression from adenomas to CRCs. Since we did not observe significant difference in the expression of any of the examined genes between early-stage and late-stage cancers, it would be interesting to demonstrate whether the Notch pathway gets activated during the progression from colorectal adenomas to cancers in a larger cohort. To our knowledge, this is the first study investigating the mRNA expression of 13 important Notch signaling members that include 4 receptors, 5 ligands, and 4 target genes in  adenomas and CRCs using highly specific TaqMan assays in Saudi Arabian patients.
In conclusion, our study emphasizes a significant role of the Notch signaling pathway in colorectal cancers and suggests its involvement in prognosis. Hence, targeting this pathway either alone or in combination with other drugs may be a promising strategy in the management of colorectal cancers for better patients' survival outcome.

Data Availability
All data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest in this work.