*2.1.1. LGR5*

Gastric cancer accounts for around 10% of all new cancers (one million per year), and it is the second leading cause of cancer death globally (700,000 deaths per year). The prognosis of CG is very poor, with a survival rate below 30% at 5 years post diagnosis [1, 2]. It is usually asymptomatic or causes nonspecific symptoms at early stages. When symptoms appear, the cancer has usually reached an advanced stage and there is presence of metastasis, being this dissemination a main cause of the severe prognosis. GC-associated high mortality is the result of its silent nature and the extremely high heterogeneity exhibited between individuals and also within gastric tumors. This heterogeneity involves, at the molecular level, a broad variety of gene mutations, amplifications and/or expression alterations, diverse DNA methylation profiles and differences in the activation or inactivation of particular signaling pathways. Thus, in the last years there has been substantial progress in the elucidation of the genomic landscape of GC due to advances in high-throughput technologies and the effort of international consortiums. Consequently, gastric cancer has been recently reclassified and stratified into several distinct subtypes based on molecular and genetic/epigenetic alterations [5, 6]. In particular, GCs have been classified according to defined genetic signatures, the status of *TP53* and the presence of microsatellite instability [5, 6]. Importantly, the heterogeneity in GC involves critical consequences in terms of differential response to therapy, resistance and recurrence [6]. Nevertheless, current therapeutic strategies in GC are not adapted to GC heterogeneity and depend on the stage of the tumor. Clinically, first-line treatment consists of surgical resection (except in cases with advanced metastasis), followed by chemotherapy with cytostatic agents such as cisplatin, 5-fluorouracil (5-FU), taxanes or irinotecan, or in combinations as ECF (epirubicin, cisplatin and 5-FU) and 5-FU plus docetaxel or cisplatin (or irinotecan) [1, 7]. These treatments have been internationally and generally accepted and used since last century. In the case of metastatic dissemination, patients whose tumors exhibit high levels of HER2 receptor expression also receive Trastuzumab, a monoclonal antibody against HER2. Initially, patients respond to chemotherapy, but cancer cells eventually become resistant, facilitating the occurrence of relapses. Even with the increase in survival facilitated by the incorporation of chemotherapy, the median overall survival of patients with CG remains

low, being one of the survivals associated with cancer lower.

improve the prognosis of patients.

44 Gastric Cancer

**2. Cancer stem cells in gastric cancer**

It has been noticed that the incidence of GC has declined over time mostly in developed countries, due to improving living standards. However, and despite increasing knowledge and improvements in the standard of care, therapy resistance and metastasis remain the main causes of treatment failure and death in GC patients and GC as a disease remains a serious and significant social concern. Consequently, identifying the major GC drivers and the molecular and cellular mechanisms responsible for the GC heterogeneity and maintenance is crucial to understand the pathobiology of GC and establish optimal therapies that able to

Several types of solid cancers, including gastric cancers, contain phenotypically and functionally heterogeneous cancer cells [8]. These cancers present a small subpopulation of cells that display The human leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a member of the G protein-coupled transmembrane receptor (GPCR) superfamily. LGR5 is a receptor for R-spondins that belong to the WNT signalling complex at the membrane level [15] and is also a target gene of this pathway [16]. LGR5 is overexpressed in a variety of human cancers, including tumors of the digestive tract such as colorectal [17] or gastric cancer [18–20], wherein it has been postulated as a CSC marker. LGR5 is an established stem cell marker of the intestine, and several studies on mice and humans have shown that LGR5-positive stem cells are the cells-of-origin of intestinal and colorectal cancer [21–27]. In the stomach, there are also increasing evidence postulating LGR5 as a stem cell and CSC marker. Thereby, LGR5 expression in gastric mucosa is almost restricted to a subset of cells located at the base of the pyloric glands, distribution that fits well with the multiple sites of the gastric cancer in humans [28]. Through *in vivo* lineage tracing experiments, these authors found that LGR5-positive cells were self-renewing and multipotent and were responsible for the renewal of the gastric epithelium. Interestingly, they observed that the transformation of this population of stem cells could drive gastric tumorigenesis *in vivo* [28], fact that has been strongly demonstrated more recently by Li and collaborators [29]. Consistently with the observation in mice, in human gastric tissue, LGR5 expression has also been found in the bottom of gastric glands [30]. Supporting the putative role of LGR5 as a gastric CSC regulator in humans, independent studies have reported LGR5 overexpression in human gastric cancer samples respect to normal gastric mucosa in a progressively increasing manner from well-differentiated to poorly differentiated gastric carcinomas [20, 31]. Furthermore, LGR5 expression has been strongly linked to a high degree of tumor infiltration, high TNM stage, recurrence and dismal prognosis of gastric cancer patients [18–20, 31]. More recently, Wang and collaborators have shown that sphere cells derived from a gastric cancer cell line presented increased expression of some canonical stem regulators, being LGR5 particularly elevated [32]. They also showed that ectopic LGR5 overexpression potentiated the sphere cell growth and cell migration capabilities of gastric cancer cells and also their tolerance to oxaliplatin, associating LGR5 expression with the characteristic features of CSCs [32]. These findings are in concordance with previous observations showing the impairment of the invasiveness and the reduction in the expression of metalloproteinase 2 (MMP2) and β-catenin in gastric cancer cells in response to LGR5 silencing *in vitro*; and revealing a positive correlation between the expression of LGR5 and MMP2 in gastric cancer tissue samples [20]. Regarding the implications of the carcinogenic agent *H. pylori* in the LGR5 positive cells in the stomach, it has been found that this population of cells is expanded in gastric cancer tissues affected by the bacteria, indicating that LGR5 likely represents a marker of stem cells susceptible of oncogenic transformation driven by *H. pylori* [13, 33].

#### *2.1.2. CD133*

CD133 (also Prominin 1) is a pentaspan transmembrane glycoprotein present in embryonic epithelial structures, thought to function as an organizer of plasma membrane topology, and regulating the maintenance of the appropriate lipid composition within the plasma membrane [34]. CD133 has been presented as a marker of cancer stem cells in colon, pancreas, brain or lung cancer [35], yet its role in gastric CSCs is controversial. Several findings related to different aspects of gastric CSCs have been published in support of its role as a gastric CSC marker and regulator. In gastric cancer cell lines, CD133 silencing abrogates sphere formation capacity [36] and, consistently, CD133 has been found overexpressed in gastric sphere cultures [37, 38]. Noteworthy, a large number of publications show increased CD133 expression in human gastric cancer tissue respect to non-neoplastic gastric mucosa and highlight the prognostic significance of CD133, associating its overexpression with a big plethora of adverse clinic-pathological features, such as elevated cellular proliferation rates, high T stage, venous invasion, lymph node and distant metastasis, chemoresistance, recurrence, poor 5-year disease-free and overall survival and so on [37, 39–42]. According to this, studies performed in gastric cancer cell lines demonstrate that CD133-positive gastric cancer cells present a CSC phenotype, since they are more tumorigenic, more chemoresistant and exhibit higher migration or invasion capacities than CD133-negative cells [37, 38, 43]. However, some controversial findings have been published indicating that CD133 expression is not a *sine qua non* condition for gastric cancer cells to exhibit properties of CSCs. Thus, Takaishi et al. isolated different subpopulations of cells from gastric cancer cell lines according to the expression of CD133, CD44, CD26 and other cell surface markers and showed that CD133-positive cells did not exhibit characteristics of CSCs [44]. Similar results have been obtained in other investigations using human gastric cancer specimens as source of cells, in which CD133-positive cells were not able to reproduce tumors in immunodeficient mouse models [45, 46].
