Oncogenic Role of Cancer Stem Cell LGR5 in Colorectal Cancer Progression

Department of Radiologic Technology, School of Allied Medical Sciences, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran


Introduction
. A schematic pattern of the LGR5-mediated activation of colorectal tumor growth through interaction with the WNT/β-catenin signaling pathway. Figure 2 illustrates that overexpression of the LGR5 gene can affect colorectal carcinogenesis and lead normal mucosa to advanced cancer. Although several investigations indicated the role of the LGR5 gene in CRC (Table 1), molecular mechanisms of LGR5 and CRC have been poorly understood. In this review, we discuss the oncogenic role of LGR5 in CRC progression. Real-time PCR Mouse model Shin et al. (2021) Overexpression of LGR5 protein was correlated with advanced stage, cell differentiation, depth of infiltration, poor overall survival. Metastasis, vascular invasion, liver metastasis, and LNM. LGR5 was an independent risk factor [40].
Real-time RT-PCR Primary tumors from CRC patients He et al. (2014) The expression level of LGR5 showed a poor prognosis [53]. Meta-Analysis Meta-analysis  Upregulation of LGR5 in tumors and adenomas was observed and considered as a prognostic marker for CRC [43].

Side-view confocal endomicroscopy
Mice model Choi et al. (2014) Decrease in cell proliferation, growth, migration, and invasion by knock-downing LGR5

Methods
In the current investigation, we obtained many articles published in English using electronic databases, including PubMed and Google Scholar, Scopus, and Web of Sciences. The following keywords were used in the search strategy: colorectal cancer, cancer stem cell, LGR5, and adenoma-carcinoma sequence.

Structure of LGR5
LGR5 is a member of the G protein-coupled receptors (GPCRs) family with a large extracellular domain counting multiple leucine-rich repeats (LRRs). R-spondin proteins are the biological ligands of LGR5. The LGR5 is a member of GPCR class A receptor proteins that are located on chromosome 12q22 with ~ 144kb longe [20].
LGR family proteins have been divided into three categories, including A, B, and C.
LGR5 belongs to the B receptor group and has 13-18 LRR-containing domains and a linker area between the last LLR domain and the first transmembrane [21]. Furthermore, LGR5 is a member of the Wnt signaling pathway; co-stimulation of LGR5 ligand along with R-spondin 1 and Wnt-3a increased induction of LGR5 gene expression and initiation of related signaling pathways.
LGR5 also cooperates with LRP6 and FZD5 via a clathrin-dependent pathway to form a ternary complex upon Wnt ligand binding. Although the LGR5 gene can promote different types of cancers via the regulation of Wnt and NOTCH signaling pathways, it can be specifically expressed in the small intestinal crypt and introduced as a potential marker for CRC [21,22].

The Role of LGR5 in Cancer Progression
Identifying cancer biomarkers can provide relevant and effective ideas for developing new anti-cancer treatment strategies [12]. An abnormal increase in LGR5 may be one of the most common changes at the molecular level in some human cancers, leading to long-term enhancement of Wnt / β-catenin signaling [23].
LGR5-mediated suppression of Wnt / βcatenin signaling has also been reported in certain cancers such as B cell malignancies. To date, a therapy targeting the LGR5 signaling pathway in clinical approaches is not available. However, evidence suggests that the endogenous LGR5 + cell population plays key roles in tumor onset, progression, and metastasis [24]. Thus, targeted therapeutic modulation of the LGR5 + cancer cell population by targeting Wnt/β-catenin signals through the targeted drug delivery system or genome modification may be promising for new anticancer therapies [25]. In addition, a combination of therapeutic drugs targeting both LGR5 + and LGR5 − cancer cells may receive more attention due to the plasticity of the cancer cells [13,26]. Also, targeting more cancer cells using dual biomarkers may be much safer and more effective for anticancer treatment [27]. The role of this biomarker in the treatment of cancers in other tissues of the body is also being investigated, where it is found to be more effective in identifying gastrointestinal cancers. Available evidence suggests that high-grade serous ovarian carcinoma originates most often from stem cells in the fallopian tube epithelium, and the Wnt signaling pathway augmented by LGR5 supports tumor development and progression [28]. Additionally, T regulatory cells in the immune system modulation can increase the LGR5 expression in gastric cancer cells through the TGF-β1 gene and signaling pathway. This pathway may include activation of the Wnt signaling, and the expression of this biomarker via TGF-β gives a prognosis in gastric cancer [29]. Another study has reported that suppression of this marker is implicated in the treatment or prevention of breast cancer [30]. Also, LGR5 may serve as a future potential biomarker for patient risk stratification and metastasis in papillary thyroid cancer [31]. Therefore, identifying these biomarkers in the treatment of various cancers can greatly benefit.

The Effect of LGR5 in CRC
Overexpression of the LGR5 gene in CRC patients was related to the canonical Wnt signaling activation and poor outcomes of patients [10,32]. Researchers indicated that β-catenin is induced by Wnt signaling activation, resulting in LGR5 upregulation and carcinogenesis. So, they suggested that LGR5 gene upregulation is an important factor in CRC progression [33,34]. LVI was found to be associated with malignant features, including inflammation, epithelial-mesenchymal (EMT), angiogenesis, and advanced stages [35]. Gzil et al. demonstrated that LGR5 gene expression was significantly linked to CRC patients with positive LVI and invasion; in addition, LGR5 gene expression could be able to activate some downstream signaling Wnt/β-catenin pathways, such as c-MYC, CDKN1A, and NOTCH [36]. Wang et al. reported that LGR5 overexpression was correlated with tumor size, malignant features, and Hepatocellular carcinoma progression [37]. Also, previous studies illustrated that the expression level of the LGR5 gene was a novel prognostic biomarker for CRC [10,25,38].
Recently, Shin et al. have shown that knock out of DDK2N reduced the expression level of the LGR5 gene and increased CRC progression in a mouse model. They suggested that the expression levels of LGR5 could be controlled with targeted therapy [39].
Results obtained from Xu et al. showed that upregulation of LGR5 protein in CRC tissue was linked to advanced stages, cellular differentiation, depth of infiltration, lymph nodes metastasis (LNM), vascular invasion, liver metastasis, and distant metastasis. In contrast, a significant correlation of decreased expression of LGR5 protein with better overall survival in patients with CRC was observed. Furthermore, they mentioned that LGR5 may be an independent risk factor for CRC prognosis [40].
In another study, Ihemelanda et al. revealed the prognostic role of LGR5 as a potential biomarker for the detection of CRC [41]. Jang et al. identified a significant association of the LGR5 gene and protein with clinicopathological features, including older age, welldifferentiated tumors, and nuclear β-catenin expression. Furthermore, the overexpression level of LGR5 was found during the adenoma-carcinoma sequence [42]. Choi et al. reported LGR5 upregulation in colonic tumors and its association with malignancy risk of adenomas using side-view confocal endomicroscopy in mice model. Besides, they suggested LGR5 protein as a potential biomarker for the prognosis of colon cancer [43].
Szkandera et al. found that the LGR5 rs17109924 T>C wile-type genotype was correlated with LGR5 upregulation and enhanced resistance to 5-FU-based chemotherapy. Furthermore, they suggested the LGR5 rs17109924 T>C can be a prognostic marker for time recurrence in patients with colon cancer treated with 5-FU-based chemotherapy [44].

LRG5 Targeted Therapy for CRC Treatment
Although the structural feature of LGR5 is known, there are no approved LGR5 inhibitors. Junttila et al. utilized anti-LGR5-vc-MMAE antibody-drug conjugate (ADC) for targeted therapy of CRC, where LGR5-MMAE was capable of decreasing tumor size and growth in a mouse model [45]. Also, researchers used the LGR5-iCaspase9 knock-in organoids with cetuximab (anti-EGFR antibody) and oxaliplatin (platinum-based chemotherapeutic), where the potential of combined chemotherapy potentiates LGR5 + CSCs targeting for CRC cells [46]. The functional role of miR-100 as the LGR5 gene inhibitor was demonstrated by Zhou et al., where miR-100 was found to reduce LGR5 expression in colon cancer via joining 3untranslated regions [47]. Some new methods, such as CRISPR/Cas9 could be utilized for LGR5 therapy investigations [48,49].

Conclusions
In summary, this review indicated that LGR5 gene upregulation is an independent prognostic biomarker for aggressive clinicopathological features in CRC. The high expression level of LGR5 can lead normal tissue to adenoma-carcinoma sequence; in addition, its expression was associated with chemoresistance.
LGR5 interacts with many signaling pathways like Wnt/βcatenin. An increasing body of evidence suggests an important role of LGR5 in CRC carcinogenesis. Moreover, finding the exact mechanisms of LGR5 and its effect on CRC progression needs more investigations. Therefore, the utilization of new methods for LGR5 detection and targeted therapy might become novel research approaches for CRC treatment.