*2.1.4. CD24*

CD24 encodes a cell surface sialoglycoprotein that is physiologically expressed in developing or regenerating tissues and regulates processes such as lymphocyte development [59] or neurogenesis [60]. As other stem cell genes, CD24 is expressed in hematologic malignancies and several solid tumors including gastric cancer. Suggesting a role for CD24 in gastric CSCs, some studies by using gastric cancer cell lines have shown that derived spheres are enriched in the expression of CD24 (and CD44) [51] and also that CD24 modulates positively cell migration, while its inhibition entails apoptosis [61]. However, Takaishi et al. were unable to find properties of CSCs in a CD24-positive population in terms of sphere forming capacity and tumorigenicity in mice models [44]. With regard to patients with gastric cancer, CD24 expression progressively increases in samples of normal gastric mucosa, non-atrophic chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric cancer [61]. Moreover, CD24 expression has been associated with adverse clinicopathological and prognostic aspects such as depth of tumors, lymph node status, TNM stage and reduced overall survival [62], fact that underlines its relevance in the disease.

#### *2.1.5. SOX transcription factors*

SOX factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems including the gastrointestinal tract [63]. There are at least 20 members divided into eight groups (from A to H), based on their HMG sequence identity in humans. Members within a group preserve higher than 80% identity in their HMG-domain and share other well-conserved regions. In addition, they share biochemical properties, have overlapping expression patterns and perform synergistic or redundant functions [63]. SOX proteins play critical roles during the development of several cell types and tissues in the embryo. They are also essential for stem cell types in the adult through the regulation of the cell fate determination, differentiation and proliferation [63]. SOX members fulfill their role by activating or repressing transcription and their action on target genes is context dependent, relying on other transcription factors with which they may directly interact for specificity. Dysfunction of SOX factors has been implicated in several human diseases. Such diseases are consistent with SOX function and expression pattern during embryonic development. A growing number of evidences are demonstrating that the expression and function of SOX factors are altered in a variety of cancers, and their roles in these malignancies are related to their stemness feature [64].

#### *2.1.5.1. SOX2*

gastric tumors respect to adjacent normal tissue. The authors also showed that ectopic expression of CD44v8-10, but not standard CD44, in gastric cancer cells potentiates their ability to initiate tumors in mice at limiting cell concentrations and that total CD44 silencing impairs tumor-initiating potential of cells, which could be rescued by restoration of CD44v8-10, but

CD24 encodes a cell surface sialoglycoprotein that is physiologically expressed in developing or regenerating tissues and regulates processes such as lymphocyte development [59] or neurogenesis [60]. As other stem cell genes, CD24 is expressed in hematologic malignancies and several solid tumors including gastric cancer. Suggesting a role for CD24 in gastric CSCs, some studies by using gastric cancer cell lines have shown that derived spheres are enriched in the expression of CD24 (and CD44) [51] and also that CD24 modulates positively cell migration, while its inhibition entails apoptosis [61]. However, Takaishi et al. were unable to find properties of CSCs in a CD24-positive population in terms of sphere forming capacity and tumorigenicity in mice models [44]. With regard to patients with gastric cancer, CD24 expression progressively increases in samples of normal gastric mucosa, non-atrophic chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric cancer [61]. Moreover, CD24 expression has been associated with adverse clinicopathological and prognostic aspects such as depth of tumors, lymph node status, TNM stage and reduced overall survival [62], fact that under-

SOX factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems including the gastrointestinal tract [63]. There are at least 20 members divided into eight groups (from A to H), based on their HMG sequence identity in humans. Members within a group preserve higher than 80% identity in their HMG-domain and share other well-conserved regions. In addition, they share biochemical properties, have overlapping expression patterns and perform synergistic or redundant functions [63]. SOX proteins play critical roles during the development of several cell types and tissues in the embryo. They are also essential for stem cell types in the adult through the regulation of the cell fate determination, differentiation and proliferation [63]. SOX members fulfill their role by activating or repressing transcription and their action on target genes is context dependent, relying on other transcription factors with which they may directly interact for specificity. Dysfunction of SOX factors has been implicated in several human diseases. Such diseases are consistent with SOX function and expression pattern during embryonic development. A growing number of evidences are demonstrating that the expression and function of SOX factors are altered in a variety of cancers, and their roles in these malignancies are related to their

not standard CD44, expression [46].

lines its relevance in the disease.

*2.1.5. SOX transcription factors*

stemness feature [64].

*2.1.4. CD24*

48 Gastric Cancer

SOX2 belongs to the SOXB1 subgroup along with the closely related SOX1 and SOX3. SOX2 is required for establishing embryonic stem cells and the maintenance of the early embryo [65]. It is also one of the factors necessary for reprogramming terminally differentiated cells into induced pluripotent stem cells [66]. Furthermore, SOX2 belongs to the core transcriptionally circuitry found on the regulatory regions of many genes with embryonic stem cell-specific expression [67]. This evidence demonstrates that SOX2 is a key factor in the control of embryonic stem cells fate and activity. SOX2 has additional functions during development, thus emerging as a critical regulator of stem cell maintenance and cell fate decisions. Furthermore, SOX2 also plays a relevant role during adulthood controlling tissue homeostasis and regeneration. Its expression is elevated in different populations of stem cells [68–71], and its high levels can be used to identify quiescent stem cells and distinguish them from transient amplifying progenitors [72, 73]. SOX2 is a regulator of gastric stem cells highly relevant for gastric patterning during development [74] and involved in the physiological renewal of the gastric epithelium in the adulthood [71, 75]. SOX2 displays several roles in cancer as an oncogenic driver, prognostic factor or a marker and regulator of CSCs [76–80]. In GC, its action is controversial. Several authors observed that SOX2 is frequently downregulated in gastric cancer [81–86]. Furthermore, low SOX2 expression is associated with shorter survival time [82] and also with worse prognosis [84]. In contrast, higher SOX2 levels are found among patients who have better prognosis [84]. In a large set of patients, Wang and coworkers demonstrated that SOX2 expression is progressively reduced during gastric carcinogenesis, from normal into invasive cancers including a series of premalignant states, supporting the role of SOX2 decrease as a robust predictor of disease outcome [85]. Similarly, SOX2 downregulation is linked with diffuse type of cancer with SOX2 expression becoming a good biomarker to discriminate between tumor (negative) and non-tumor (positive expression) and also high/low grades of tumor malignancy [86]. The regulation of SOX2 expression in GC has been mostly associated to epigenetic changes. Thus, aberrant DNA methylation has been shown as a key mechanism underlying SOX2 downregulation in a set of primary gastric carcinoma samples [82]. Besides promoter methylation, miR-126 overexpression also decreases SOX2 levels and therefore acts as a tumor suppressor [83]. Recently, it has been shown that SOX2 has an important role in gastric differentiation [87]. It is known that during gastric carcinogenesis, the homeobox transcription factor CDX2 is critical for intestinal differentiation driving the onset of intestinal metaplasia (IM) [88, 89]. Thereafter, Camilo and coworkers showed that SOX2 is associated with gastric differentiation in incomplete IM and is lost in the progression to dysplasia, whereas CDX2 is acquired *de novo* in IM and maintained in dysplasia [87]. Taken it into account, the authors hypothesized that balance between gastric and intestinal differentiation programs might interfere on the gastric carcinogenesis progression [87]. Since SOX2 and CDX2 expression were found in about half of the cases, the interaction of both transcription factors in gastric carcinogenesis remains to be investigated. Functional characterization performed in gastric epithelial cell lines showed that SOX2 ectopic activation inhibits cell proliferation through G1 cell-cycle arrest and induces apoptosis by decreasing cyclin D1 and phosphorylated Rb and increasing p27Kip1 protein levels [82]. Overall, the authors observed that SOX2 performs a critical part in gastric carcinogenesis, operating as a tumor suppressor. Similarly, Wang and coworkers verified that enforced SOX2 expression inhibited proliferation, increased apoptosis and reduced invasion and motility, both in vitro and in vivo [85]. Mechanistically, SOX2 directly transactivates PTEN. Therefore, this SOX2-dependent PTEN upregulation may directly orchestrate downstream phospho-Akt dephosphorylation, affecting diverse cellular phenotypes such as survival, growth, proliferation and migration [85]. These studies show that SOX2 plays important roles in gastric epithelial cells growth inhibition through cell-cycle arrest and apoptosis [90]. Regarding its relationship with *H. pylori*, SOX2 expression is decreased by the bacteria, and this inhibition leads to an upregulation of CDX2 expression [75, 91, 92]. Additionally, *in vitro* and in a mice model infected with *Helicobacter* spp. demonstrated that CDX2 and SOX2 are downstream targets of the BMP (bone morphogenetic protein) pathway in gastric carcinogenesis. The authors showed that *H. pylori* upregulates BMP pathway, through an increase in BMP2, SMAD4 and pSMAD1/5/8 expression. Thus, SOX2 expression was downregulated by *H. pylori* and the BMP pathway [93]. From a mechanistic perspective, it was postulated that the activation of an intestinal differentiation program may occur concomitantly with the silencing of a gastric differentiation, induced or controlled by SOX2 [93]. Another recent study identified that the bacteria might trigger its pro-carcinogenic activity through a blockage of SOX2 [85]. However, other authors verified that overexpression of SOX2 is associated with tumor invasion, lymph node metastasis and chemoresistance [94–97]. Tian and coworkers were able to show that SOX2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer, suggesting an oncogenic effect of SOX2 in the stomach [94]. In addition, it has been demonstrated that SOX2 overexpression was significantly correlated with lymph node metastasis and the stage of tumor invasion in gastric cancer indicating that SOX2 might be a predictive prognostic factor [95]. Hutz and coworkers proved that high levels of SOX2 are involved in gastric carcinogenesis by regulating the expression of genes associated with proliferation, apoptosis and cell cycle regulation, *in vitro* and *in vivo* [96]. Functionally, the SOX2 suppression induced a decrease in cell proliferation, which coincided with an increase of apoptosis in AZ-521 cells. Similarly, blocking of SOX2 in a xenograft mouse model resulted in reduced tumor growth [96]. Moreover, the expression of SOX2 in human gastric tumor samples was observed at high proliferation rate sites [96]. Likewise, SOX2 overexpression in gastric cancer has been recently observed in other study, where the surge in the expression is attributed to SOX2 locus copy number variation, being related as well with the presence of regional lymph node metastases [98].

#### *2.1.5.2. SOX9*

SOX9 is overexpressed in a variety of human cancers, being its high levels correlated with malignant character and progression in prostate, lung, breast and brain tumors [80, 99, 100]. SOX9 expression is also elevated in tumors of the digestive system such as esophageal, colorectal and pancreatic cancers [101, 102]. In esophageal and pancreatic tumors, SOX9 stimulate self-renewal properties [102, 103]. However, in colorectal cancer, there are contradictory results between functional studies and clinical samples, suggesting a context-dependent activity of SOX9 [100, 104]. Remarkably, several studies have reported clinical implications of SOX9 in GC. Thereby, in GC patients, high tumoral SOX9 expression has been observed and associated with advanced TNM stages and lower overall patient survival [105]. Interestingly, in clinical samples, high levels of SOX9 correlate with elevated expression of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) [106], which facilitates GC metastasis, and are positively associated with lymph nodes metastasis and advanced TNM stage [107]. In samples from patients, there is also an inverse relation between SOX9 and the tumor suppressor gastrokine 1 (GKN1), relationship also observed in GC cell lines, wherein GKN1 negatively regulates SOX9 expression [106, 108]. Furthermore, elevated SOX9 expression in gastric tumors is associated with the activation of the WNT canonical oncogenic pathway, with whom it establishes a feedback regulatory loop [105].

Similarly, Wang and coworkers verified that enforced SOX2 expression inhibited proliferation, increased apoptosis and reduced invasion and motility, both in vitro and in vivo [85]. Mechanistically, SOX2 directly transactivates PTEN. Therefore, this SOX2-dependent PTEN upregulation may directly orchestrate downstream phospho-Akt dephosphorylation, affecting diverse cellular phenotypes such as survival, growth, proliferation and migration [85]. These studies show that SOX2 plays important roles in gastric epithelial cells growth inhibition through cell-cycle arrest and apoptosis [90]. Regarding its relationship with *H. pylori*, SOX2 expression is decreased by the bacteria, and this inhibition leads to an upregulation of CDX2 expression [75, 91, 92]. Additionally, *in vitro* and in a mice model infected with *Helicobacter* spp. demonstrated that CDX2 and SOX2 are downstream targets of the BMP (bone morphogenetic protein) pathway in gastric carcinogenesis. The authors showed that *H. pylori* upregulates BMP pathway, through an increase in BMP2, SMAD4 and pSMAD1/5/8 expression. Thus, SOX2 expression was downregulated by *H. pylori* and the BMP pathway [93]. From a mechanistic perspective, it was postulated that the activation of an intestinal differentiation program may occur concomitantly with the silencing of a gastric differentiation, induced or controlled by SOX2 [93]. Another recent study identified that the bacteria might trigger its pro-carcinogenic activity through a blockage of SOX2 [85]. However, other authors verified that overexpression of SOX2 is associated with tumor invasion, lymph node metastasis and chemoresistance [94–97]. Tian and coworkers were able to show that SOX2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer, suggesting an oncogenic effect of SOX2 in the stomach [94]. In addition, it has been demonstrated that SOX2 overexpression was significantly correlated with lymph node metastasis and the stage of tumor invasion in gastric cancer indicating that SOX2 might be a predictive prognostic factor [95]. Hutz and coworkers proved that high levels of SOX2 are involved in gastric carcinogenesis by regulating the expression of genes associated with proliferation, apoptosis and cell cycle regulation, *in vitro* and *in vivo* [96]. Functionally, the SOX2 suppression induced a decrease in cell proliferation, which coincided with an increase of apoptosis in AZ-521 cells. Similarly, blocking of SOX2 in a xenograft mouse model resulted in reduced tumor growth [96]. Moreover, the expression of SOX2 in human gastric tumor samples was observed at high proliferation rate sites [96]. Likewise, SOX2 overexpression in gastric cancer has been recently observed in other study, where the surge in the expression is attributed to SOX2 locus copy number variation, being related as well with the

SOX9 is overexpressed in a variety of human cancers, being its high levels correlated with malignant character and progression in prostate, lung, breast and brain tumors [80, 99, 100]. SOX9 expression is also elevated in tumors of the digestive system such as esophageal, colorectal and pancreatic cancers [101, 102]. In esophageal and pancreatic tumors, SOX9 stimulate self-renewal properties [102, 103]. However, in colorectal cancer, there are contradictory results between functional studies and clinical samples, suggesting a context-dependent activity of SOX9 [100, 104]. Remarkably, several studies have reported clinical implications of SOX9 in GC. Thereby, in GC patients, high tumoral SOX9 expression has been observed and associated with advanced TNM stages and lower overall patient survival [105]. Interestingly, in clinical

presence of regional lymph node metastases [98].

*2.1.5.2. SOX9*

50 Gastric Cancer

Noteworthy, SOX9 is a critical executor of the carcinogenic action of *H. pylori*. According to this notion, the bacterium induces SOX9 expression in pre-tumorigenic gastric mouse cells [109] and also in GC cells [105], being the induction more pronounced in response to specimens of *H. pylori* containing the pathogenically significant *CagA* virulence factor. Notably, SOX9 is required for bacteria-induced GC cell proliferation, induction of β-catenin and acquisition of stem cell-like properties. Mechanistically, it has been found that TNFα and IL-1β cytokines, involved in the inflammatory response to *H. pylori*, induce the expression of SOX9 in mouse models and GC cells [105, 109], being probably the action of TNFα in human GC cells stronger and more extensive. In fact, TNFα high levels correlated with SOX9 upregulation in *H. pylori*-positive GC samples, and there was a positive association between them in two independent large cohorts of GC samples [TCGA and ACRG] [105]. Overall, these results identified a novel association between SOX9 and *IL1-β* and *TNFα* cytokines, which links *H. pylori* infection with SOX9 and GC outcome in patients, evidence supported by other studies [110–114]. SOX9 represents a key driver of GC and given the importance of its strong clinical implications, elucidating the molecular mechanism of its action in GC has constituted an important challenge to identify novel and suitable therapeutic targets. With respect to that, it is known that SOX9 establishes a feedback regulatory loop with WNT/β-catenin signaling pathway. Consistently, SOX9 abrogation in GC cells diminishes *CYCLIN D1* and *c-MYC* expression, and there is a positive correlation between these genes and SOX9 in patient samples [105]. Functionally, SOX9 silencing in GC cells promotes apoptosis and senescence through BMI1 decline and the consequent upregulation of p21CIP [105]. SOX9 silencing also supposes detrimental effects on the subpopulation of gCSCs, reflected by a reduction in tumorsphere self-renewal and decreased tumor initiating capability [105]. Paralleling these effects, and likely due to its functions in gCSCs, SOX9 mediates cisplatin chemoresistance [105, 114], fact that might explain the reduced disease-free survival of patients presenting tumors with high *SOX9* expression levels [105]. Additionally, there are other SOX members associated to GC. Thus, SOX4 has been shown to display pro-oncogenic activities and become upregulated with gastric cancer progression, in the population of gCSCs and in response to *H. pylori* infection [115–117]. Finally, SOX18 mRNA levels are increased in gastric cancer tissues compared to normal tissue, and the frequencies of both lymphovascular invasion and lymph node metastases are higher in SOX18 positive than in the negative group. Furthermore, both the 5-year survival and the recurrence-free survival were shorter for SOX18-positive cancers suggesting that SOX18 expression might be a prognostic tumor marker and a potential therapeutic target in gastric cancer [118].
