Rac1, a Rho GTPase family member, is dysregulated in a variety of tumor types including gastric adenocarcinoma, but little is known about its role in cancer stem-like cells (CSCs). Therefore, Rac1 activity and inhibition were examined in gastric adenocarcinoma cells and mouse xenograft models for epithelial-to-mesenchymal transition (EMT) and CSC phenotypes. Rac1 activity was significantly higher in spheroid-forming or CD44+ gastric adenocarcinoma CSCs compared with unselected cells. Rac1 inhibition using Rac1 shRNA or a Rac1 inhibitor (NSC23766) decreased expression of the self-renewal transcription factor, Sox-2, decreased spheroid formation by 78%–81%, and prevented tumor initiation in immunodeficient mice. Gastric adenocarcinoma CSCs had increased expression of the EMT transcription factor Slug, 4.4- to 8.3-fold greater migration, and 4.2- to 12.6-fold greater invasion than unselected cells, and these increases could be blocked completely with Rac1 inhibition. Gastric adenocarcinoma spheroid cells were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed with Rac1 shRNA or NSC23766. The PI3K/Akt pathway may be upstream of Rac1, and JNK may be downstream of Rac1. In the MKN-45 xenograft model, cisplatin inhibited tumor growth by 50%, Rac1 inhibition by 35%, and the combination by 77%. Higher Rac1 activity, in clinical specimens from gastric adenocarcinoma patients who underwent potentially curative surgery, correlated with significantly worse survival (P = 0.017). In conclusion, Rac1 promotes the EMT program in gastric adenocarcinoma and the acquisition of a CSC state. Rac1 inhibition in gastric adenocarcinoma cells blocks EMT and CSC phenotypes, and thus may prevent metastasis and augment chemotherapy.

Implications: In gastric adenocarcinoma, therapeutic targeting of the Rac1 pathway may prevent or reverse EMT and CSC phenotypes that drive tumor progression, metastasis, and chemotherapy resistance. Mol Cancer Res; 15(8); 1106–16. ©2017 AACR.

There are nearly one million new gastric cancer cases worldwide per year and nearly 700,000 gastric cancer–related deaths per year, thus accounting for almost 10% of all cancer-related deaths (1). The majority of patients with gastric adenocarcinoma present with locally advanced or metastatic disease. Overall survival for patients with metastatic disease is 3–5 months with best supportive care (2). The response rate to multiagent chemotherapy can be 50% or greater, but nearly all patients develop chemotherapy resistance, and median survival is extended only to 9–11 months (3).

The cancer stem cell (CSC) theory states that cancers harbor a subset of cells that share characteristics of normal stem cells such as the capacity for self-renewal and differentiation (4).These purported CSCs are more resistant to chemotherapy than non-CSCs and may be the source of metastases (5). Methods to identify CSCs include tumor formation in immunodeficient mice, spheroid colony formation in vitro, and expression of certain cell surface markers. In one study, potential CSC markers were examined in six gastric cancer cell lines, and CD44 was the only CSC marker associated with spheroid colony formation in vitro and tumor formation in immunodeficient mice (6).

Epithelial-to-mesenchymal transition (EMT) is a biologic process by which epithelial cells lose their cell polarity and cell-to-cell adhesion and assume a mesenchymal phenotype which includes migratory and invasive properties (7). EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation as well as physiologic and pathologic processes, such as wound healing and the initiation of metastasis for cancer progression (8). Several transcription factors drive EMT including Snail, Slug, and Zeb1, and loss of cell surface E-cadherin expression and gain of N-cadherin expression is characteristic of EMT (9).

Several studies have found frequent mutations in RHOA in Lauren diffuse-type gastric adenocarcinoma (10–12). RhoA is the founding member of the Rho GTPase family that also includes Cdc42 and Rac1 (13). These proteins serve as intracellular molecular switches cycling between a GTP-bound active form and a GDP-bound inactive form. We recently found that RhoA signaling promotes chemotherapy resistance in diffuse-type gastric adenocarcinoma cells, and RhoA inhibition could reverse this chemotherapy resistance (14).

Building on our studies examining RhoA, for this study, we chose to examine another Rho GTPase family member, Rac1. When expression of seven Rho GTPase family members were examined in 53 gastric adenocarcinoma patient tumors and 7 gastric adenocarcinoma cell lines, only Rac1 and RhoA were found to be consistently increased (15). The Rho GTPase family of proteins regulates a diverse array of cellular processes including cell proliferation, metabolism, cytoskeletal reorganization, and activation of protein kinases (16). Rac1 appears to be deregulated in a variety of tumor types, and Rac1 hyperactivation and overexpression seems to correlate well with aggressive growth and other malignant characteristics (17). There are very few studies examining the role of Rac1 in CSCs and EMT (18, 19), and no studies examining Rac1 specifically in gastric adenocarcinoma CSCs. In this study, we discover a critical role for Rac1 in promoting the EMT program in gastric adenocarcinoma cells and acquisition of a CSC state.

Cell lines and reagents

AGS, NCI-N87, and MKN-45 cells were obtained from the ATCC. SNU-668 cells were obtained from the Korean Cell Line Bank (KCLB). AGS, MKN-45, and SNU-668 cells were maintained in RPMI1640. NCI-N87 gastric cancer cell line was maintained in DMEM/F12. All media were supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mmol/L l-glutamine (“regular media”). Cancer cell lines were actively passaged for less than 6 months from the time that they were received from ATCC and United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines were followed (20). 5-Fluorouracil was purchased from US Biological, and cisplatin was purchased from Enzo Life Sciences. PI3K inhibitor (LY294002), JNK inhibitor (SP600125), ERK inhibitor (U0126), and p38 MAPK inhibitor SB203580 were purchased from Calbiochem. Rac1 inhibitor (NSC23766) was purchased from Santa Cruz Biotechnology. 5-Fluorouracil was purchased from US Biologicals, and cisplatin was purchased from Enzo Life Sciences. Gastric cancer cell lines were grown as spheroids and spheroids were counted as described previously (14).

Western blot analysis

Western blot analysis was performed as described previously (14) using the following antibodies: Akt (sc-8312, Santa Cruz Biotechnology), ERK1 (sc-271270, Santa Cruz Biotechnology), p38 (sc-81621, Santa Cruz Biotechnology), JNK2 (sc-827, Santa Cruz Biotechnology), CD24 (sc-11406, Santa Cruz Biotechnology), c-Myc (sc-40, Santa Cruz Biotechnology), Sox2 (#2748, #3579), Oct-4 (#2750), Nanog (#4893), Slug (#9585), Snail (#3879), phospho-Akt (ser473) (#9271), phospho-p38 (#9211), phospho-ERK1/2 (#9101), phospho-JNK1/2 (#9251), CD44 (#3578), Musashi-1 (#2154), cleaved caspase-3 (#9661), Rac1 (BD 61065, BD Biosciences), N-cadherin (BD 610920, BD Biosciences), Zeb1 (NBP-1-05987, Novus Biologicals), CD133 (MBS462020, MyBioSource), and β-actin (A5228, Sigma)

Rac1 GTPase activity assays

Rac1 activity assays were performed using Rac1 assay reagent (GST-PAK1-PBD on glutathione beads) according to the manufacturer's instructions (Active Rac1 Activation Assay kit, Cell Signaling Technology). RhoA activity assays were performed using Rho activation assay beads (GST-Rhotekin-RBD on glutathione beads) according to the manufacturer's instructions (Active Rho Detection kit, Cell Signaling Technology). Cdc42 activity assay was performed using active Cdc42 detection kit according to the manufacturer's instructions (Active Cdc42 detection kit, Cell Signaling Technology).

FACS

Cells were dissociated using Accutase and resuspended in PBS containing 0.5% BSA. The cells were stained with FITC-conjugated CD44 (BD555478) or isotype control antibody (BD555742) from BD Biosciences on ice for 30 minutes. Cells were then washed with PBS and analyzed on a BD FACSCalibur (BD Biosciences) using Cell Quest software.

shRNA

Silencing of Rac1 was achieved via lentiviral transduction of human Rac1 shRNA (sc-36351-V; Santa Cruz Biotechnology). A scramble shRNA control (SC-108080) and a GFP control (sc108084) were also used. Maximal knockdown of Rac1 occurred 72 to 96 hours after transduction.

In vitro assays

Cancer cell proliferation, migration, invasion, and single-cell assays were performed as described previously (14).

Immunocytochemistry

Spheroids were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Following cell fixation, cells were incubated with CD44 FITC Conjugate, Sox2, Oct-4, Nanog, c-Myc, Slug, and/or Rac1 antibody in a solution of PBS with 1% BSA and 0.1% Triton X-100 at 4°C overnight. Staining was visualized using anti-mouse Alexa Flour 594 (A11005; Life Technologies) and anti-rabbit Alexa Flour 594 (A11012; Life Technologies). Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Stained cells were visualized with an inverted confocal microscope. Image processing was performed using Imaris 7.6.

IHC and immunofluorescence

IHC and immunofluorescence were performed as described previously (14). Antibodies used for IHC were anti-Rac1 (BD 610650, BD Transduction Laboratories), anti-phospho-Rac1 (#44-214G, Life Technologies), anti-phospho-JNK (#4668, Cell Signaling Technology), anti-Sox2 (#14962, Cell Signaling Technology), anti-PCNA (sc-56, Santa Cruz Biotechnology), and anti-cleaved caspase-3 (#9661, Cell Signaling Technology). To quantify IHC staining, images were digitally scanned with Panoramic Flash 250 (3DHistech) using 20×/0.8NA objective. Stained tissues were counted in five microscopic fields. The analysis was performed in Imaris 7.6 (Bitplane). Phospho-Rac1 stain was predominantly cytosol. Phospho-Rac1 scores (0–300) were calculated by multiplying the staining intensity (0, 1, 2, or 3) by the staining extent (0%–100%).

For immunofluorescence, antibodies used were anti-Rac1 (BD 610650, BD Transduction Laboratories), anti-CD44 (#5640, Cell Signaling Technology), anti-Sox2 (#3579, Cell Signaling Technology), anti-Oct-4 (#83932, Cell Signaling Technology), anti-Nanog (#8822, Cell Signaling Technology), anti-c-Myc (sc-40, Santa Cruz Biotechnology), anti-N-cadherin (BD 610920, BD Transduction Laboratories), and anti-Slug (#9585, Cell Signaling Technology).

Mouse studies

All mouse protocols were approved by the Institutional Animal Care and Use Committee. To generate subcutaneous flank tumors, 5 × 106 MKN-45 cells were resuspended in 100 μL of Hank balanced salt solution (HBSS) and injected subcutaneously into the right flank of athymic, nude, 6- to 8-week-old male BALB/c nu/nu mice (Taconic) following isoflurane anesthesia. Mice were assigned into treatment groups (5 mice per group) when tumors reached 50–100 mm3 in volume, designated as day 0. Cisplatin 2 mg/kg or carrier (PBS) was injected intraperitoneally once per week. Tumors were measured three times per week for 2 weeks, and tumor volume (TV) was calculated by using the following formula: TV = length × (width)2 × 0.52. After mice were sacrificed, tumors were excised and cut into thirds. Portions of each tumor was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and processed into 5-μm sections.

To assess for tumor initiation in vivo, 5,000 or 2 × 104 MKN-45 cells were resuspended in 100 μL of Hank balanced salt solution (HBSS) and injected subcutaneously into the right flank of athymic, Rag2/γC double knockout mice (Taconic) following isoflurane anesthesia (6). Mice were monitored weekly for tumor growth for up to 8 weeks.

Patients

Patients with adenocarcinomas arising in the stomach or gastroesophageal junction (Siewert type II or III) who underwent radical gastrectomy or esophagogastrectomy with potentially curative intent (R0 and R1) from May 2006 to March 2012 at Memorial Sloan Kettering Cancer Center (MSKCC, New York, NY) were included. The MSKCC Institutional Review Board approved this study, and informed consent for study of tumor tissue was obtained preoperatively from all patients. Tumor staging was determined from the surgical specimen and was based on the 7th edition of American Joint Committee on Cancer TNM staging system (18).

Statistical analysis

Data are represented as mean ± SD unless otherwise noted. Groups were compared using Instant 3.10 software (GraphPad). P values were calculated using Student t test. For comparisons between more than 2 groups, treatment groups were compared with the control group using one-way ANOVA with Bonferroni adjustment for multiple comparisons. For human data analyses, the continuous values are expressed as mean ± SD and analyzed using the Student t test. The categorical variables are analyzed using χ2 or Fisher exact test. Overall survival curves were plotted by the Kaplan–Meier methods and compared using the log-rank test. Cox proportional hazards regression modeling was used to examine the effect of Rac1 activity on survival while controlling for confounding covariates. Analyses were performed using IBM SPSS software for Windows version 21. A P value of less than 0.05 was considered statistically significant.

Rac1 activity is increased in gastric adenocarcinoma spheroid cells and CD44+cells

Levels of total and active Rac1 along with levels of total and active RhoA and Cdc42 were examined by Western blot analysis in four Lauren intestinal and four Lauren diffuse gastric adenocarcinoma cell lines. Levels of total Rac1, total RhoA, and total Cdc42 were relatively equal in all cell lines (Fig. 1A). Active Rac1 levels varied among the eight cell lines while active RhoA and active Cdc421 tended to be higher in diffuse cell lines compared with intestinal cell lines. Growth of cell lines in spheroid formation conditions selects for cells with CSC properties (21). Three cell lines, AGS, MKN-45, and NCI-N87 were grown as monolayers or as spheroids, and levels of active Rac1 were highly upregulated in all three cell lines when grown as spheroids (Fig. 1B). Levels of total Rac1 were the same in monolayer and spheroid cells. Takaishi and colleagues examined six gastric adenocarcinoma cell lines and found the cell surface protein CD44 to be the only gastric CSC marker associated with tumor formation in immunodeficient mice and spheroid formation in all six cell lines (22). We confirmed that AGS, MKN-45, and NCI-N87 cells grown as spheroids had increased expression of CD44 while levels of other putative gastric CSC markers such as CD133, CD24, CD133, and Musashi-1 were not reliably increased (Supplementary Fig. S1A). Cell lines were sorted by FACS for CD44 expression, and active Rac1 was found to be highly upregulated in CD44+ cells compared with CD44 cells (Fig. 1C).

Figure 1.

Rac1 activity in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of active and total Rac1, RhoA, and Cdc42 in eight gastric adenocarcinoma cell lines. Western blot analysis of active and total Rac1 in three gastric adenocarcinoma cell lines grown as monolayers and as spheroids (B) or following FACS for CD44 and CD44+ cells (C). D, Western blot analysis for total Rac1 and putative CSC markers in AGS, MKN-45, and NCI-N87 spheroid cells following transduction with Rac1 shRNA (sh.Rac1) or scrambled control shRNA (sh.Scr). E, Immunofluorescence of AGS spheroids for DAPI (blue), Rac1 (red) and CD44 (green). F, Photos and graph of AGS, MKN-45, and MNI-N87 cells following transduction with sh.Scr or sh. Rac1 and grown in spheroid formation conditions. Bars represent SD. *, P < 0.05 compared with control.

Figure 1.

Rac1 activity in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of active and total Rac1, RhoA, and Cdc42 in eight gastric adenocarcinoma cell lines. Western blot analysis of active and total Rac1 in three gastric adenocarcinoma cell lines grown as monolayers and as spheroids (B) or following FACS for CD44 and CD44+ cells (C). D, Western blot analysis for total Rac1 and putative CSC markers in AGS, MKN-45, and NCI-N87 spheroid cells following transduction with Rac1 shRNA (sh.Rac1) or scrambled control shRNA (sh.Scr). E, Immunofluorescence of AGS spheroids for DAPI (blue), Rac1 (red) and CD44 (green). F, Photos and graph of AGS, MKN-45, and MNI-N87 cells following transduction with sh.Scr or sh. Rac1 and grown in spheroid formation conditions. Bars represent SD. *, P < 0.05 compared with control.

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Next, cell lines were transduced with Rac1 shRNA or scrambled control shRNA and grown under spheroid formation conditions. Rac1 knockdown in AGS spheroids was confirmed by immunofluorescence and Western blot analysis, and also led to decreased expression of CD44 but no changes in expression of CD133, CD24, or Musashi-1 (Fig. 1D and E). Similar results were seen with Rac1 knockdown in MKN-45 and NCI-87 cells (data not shown). Rac1 knockdown dramatically reduced the ability of AGS, MKN-45, and NCI-87 cells to form spheroids by 71%–77% (Fig. 1F). NSC23766 is a chemical inhibitor of the binding and activation of Rac1 GTPase, although it is not entirely specific for Rac1 (23, 24). NSC23766 at a dose of 100 μmol/L completely abolished Rac1 activity in AGS cells grown as spheroids (Supplementary Fig. S1B) and decreased the ability of CD44+ AGS cells to form colonies from single cells by 65% at 10 days (Supplementary Fig. S1C). These data suggest that Rac1 activity in gastric cancer cells promotes spheroid formation and may play an important role in other gastric CSC phenotypes.

Activation of Rac1 is critical for the self-renewal in gastric adenocarcinoma CSCs

To further examine the involvement of Rac1 in regulating stemness in gastric adenocarcinoma cells, we next investigated expression of self-renewal proteins in AGS, NCI-N87, and MKN-45 cell lines grown as monolayers or as spheroids. We found increases in the self-renewal proteins Sox2 and Nanog, but not in Oct-4 or c-Myc, when cells were grown as spheroids (Fig. 2A). Levels of Sox 2 and Nanog were further increased in CD44+ cells following FACS (Supplementary Fig. S2A). Rac1 inhibition with Rac1 shRNA or the Rac1 inhibitor NSC23766 dramatically decreased Sox2 levels and had minor or no effects on Oct4 and Nanog levels (Fig. 2B; Supplementary Fig. S2B). Immunofluorescence analysis also confirmed decreased expression of CD44 and Sox2 in spheroid cells following transduction with Rac1 shRNA or treatment with NSC23766 (Fig. 2C). Rac1 shRNA also dramatically reduced the ability of gastric cancer cells to form colonies from single cells (Fig. 2D). Rac1 inhibition with NSC23766 had a similar effect in this single-cell assay (Supplementary Fig. S2C). Finally for MKN-45 cells, we implanted 5,000 or 2 × 104 CD133+ cells transduced with Rac1 shRNA or control shRNA into the flanks of athymic, Rag2/γC double knockout mice. All four mice implanted with MKN-45 cells treated with control shRNA grew flank tumors while none of the four mice implanted with MKN-45 cells transduced with Rac1 shRNA grew flank tumors. (Fig. 2E). Taken together, these results suggest that Rac1 activity is associated with expression of some genes related with stemness and promotes additional CSC properties such as formation of colonies from single cells and tumor formation in immunodeficient mice.

Figure 2.

Rac1 promotes Sox2 expression and self-renewal in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of self-renewal proteins for Sox2, Oct-4, Nanog, and c-Myc in three gastric adenocarcinoma cell lines grown as monolayers or as spheroids. Western blot analysis for Sox2, Oct-4, Nanog, and c-Myc in gastric adenocarcinoma cell lines grown as spheroids and transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). C, Immunofluorescence of AGS spheroids for DAPI (blue), CD44 (green), and Sox2 (red) following transduction with sh.Rac1 or sh.Scr and treatment with NSC23766 or carrier (DMSO). D, Photos from single-cell assay for AGS and MKN-45 spheroid cells following transduction with sh.Scr and sh. Rac1. E, Photos of immunodeficient mice injected with 5,000 or 2 × 104 MKN-45 spheroid cells stably transduced with sh.Scr and sh. Rac1.

Figure 2.

Rac1 promotes Sox2 expression and self-renewal in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of self-renewal proteins for Sox2, Oct-4, Nanog, and c-Myc in three gastric adenocarcinoma cell lines grown as monolayers or as spheroids. Western blot analysis for Sox2, Oct-4, Nanog, and c-Myc in gastric adenocarcinoma cell lines grown as spheroids and transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). C, Immunofluorescence of AGS spheroids for DAPI (blue), CD44 (green), and Sox2 (red) following transduction with sh.Rac1 or sh.Scr and treatment with NSC23766 or carrier (DMSO). D, Photos from single-cell assay for AGS and MKN-45 spheroid cells following transduction with sh.Scr and sh. Rac1. E, Photos of immunodeficient mice injected with 5,000 or 2 × 104 MKN-45 spheroid cells stably transduced with sh.Scr and sh. Rac1.

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PI3K/Akt may be upstream and activates Rac1, while JNK may be downstream of Rac1

Several lines of evidence indicate that the phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways play a key role in cancer stem cell biology (25–27). Gastric adenocarcinoma cell lines showed significantly higher levels of phosphorylated forms of Akt, extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase 1/2 (JNK1/2) when cells were grown as spheroids compared with when grown as monolayers (Supplementary Fig. S3A). After sorting for CD44, CD44+ cells also had more phosphorylated forms of Akt, ERK, and JNK1/2 compared with CD44 cells (Supplementary Fig. S3B). Levels of phosphorylated p38 were not changed in monolayer versus spheroids cells. To determine whether these pathways were functionally important in gastric CSCs, spheroid formation was measured after treatment with the Akt inhibitor LY294002, JKN inhibitor SP600125, ERK inhibitor U0126, or p38 inhibitor SB203580 in GFP-expressing, CD44+ spheroid cells (Supplementary Fig. S3C). The Akt inhibitor LY294002 was most effective in blocking spheroid formation followed by the JNK inhibitor SP600125 and the ERK inhibitor U0126 (Fig. 3A). The p38 inhibitor SB203580 had no effect on spheroid formation. Similar inhibitory effects on spheroid formation were seen in unselected cells treated with the Akt inhibitor or the JNK inhibitor (Supplementary Fig. S3D). These inhibitors also reduced expression of CD44 and Sox2 when measured by immunofluorescence (Supplementary Fig. S3E).

Figure 3.

PI3K/Akt pathway activates Rac1 and Rac1 activates JNK in gastric adenocarcinoma CSCs. A, Immunofluorescence photos of AGS, MKN-45, and NCI-N87 CD44+ spheroid cells treated with the Akt inhibitor LY294002, JNK inhibitor SP600125, ERK inhibitor U0126, p38 inhibitor SB203580, or carrier (DMSO). Scale bar, 50 μm. B, Western blot analysis of gastric adenocarcinoma spheroid cells for total Rac1 and total and phosphorylated MAPK proteins following stable transduction of cells with Rac1 shRNA (sh.Rac1) or control shRNA (sh.Scr). C, Western blot of gastric adenocarcinoma spheroid cells for total and phosphorylated Akt and JNK along with total and active Rac1 following treatment with the Akt inhibitor LY294002 or carrier (DMSO). Western blot analysis of gastric adenocarcinoma CD44+ spheroid cells for total and phosphorylated MAPK proteins and total and active Rac1 following treatment with the Akt inhibitor LY294002 or carrier (DMSO; D) or with the JNK inhibitor SP00125 or carrier (DMSO; E). IHC staining of gastric tumors with low or high Rac1 activity for phosphorylated Rac1, phosphorylated JNK, and Sox2. Scale bar, 20 μm.

Figure 3.

PI3K/Akt pathway activates Rac1 and Rac1 activates JNK in gastric adenocarcinoma CSCs. A, Immunofluorescence photos of AGS, MKN-45, and NCI-N87 CD44+ spheroid cells treated with the Akt inhibitor LY294002, JNK inhibitor SP600125, ERK inhibitor U0126, p38 inhibitor SB203580, or carrier (DMSO). Scale bar, 50 μm. B, Western blot analysis of gastric adenocarcinoma spheroid cells for total Rac1 and total and phosphorylated MAPK proteins following stable transduction of cells with Rac1 shRNA (sh.Rac1) or control shRNA (sh.Scr). C, Western blot of gastric adenocarcinoma spheroid cells for total and phosphorylated Akt and JNK along with total and active Rac1 following treatment with the Akt inhibitor LY294002 or carrier (DMSO). Western blot analysis of gastric adenocarcinoma CD44+ spheroid cells for total and phosphorylated MAPK proteins and total and active Rac1 following treatment with the Akt inhibitor LY294002 or carrier (DMSO; D) or with the JNK inhibitor SP00125 or carrier (DMSO; E). IHC staining of gastric tumors with low or high Rac1 activity for phosphorylated Rac1, phosphorylated JNK, and Sox2. Scale bar, 20 μm.

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We next investigated whether the PI3K/Akt and MAPK pathways are linked to Rac1 activity in gastric CSCs. Gastric cancer spheroid cells were transduced with Rac1 shRNA or scrambled control shRNA and activation of PI3K/Akt, JNK, ERK, and p38 signaling pathways was assessed. Rac1 shRNA inhibited JNK1/2 phosphorylation, but not Akt, ERK, or p38 phosphorylation (Fig. 3B). Treatment of spheroid cells with the Akt inhibitor LY294002 reduced both Rac1 activation and JNK1/2 phosphorylation (Fig. 3C). Similar results were seen when CD44+ cells rather than spheroid cells were treated with Akt or JNK inhibition (Fig. 3D). In contrast, treatment of CD44+ cells with the JNK inhibitor SP600125 did not affect levels of Rac1 activation and Akt phosphorylation (Fig. 3E). To assess whether Rac1 activity correlated with JNK pathway activation in tumors from gastric adenocarcinoma patients, we performed IHC staining for phosphorylated JNK and Sox-2 on five patient tumors with high Rac1 activity and five patient tumors with low Rac1 activity. On average, the number of cells with phosphorylated JNK and Sox 2 were 3.9-fold and 7.2-fold higher, respectively, in tumors with high Rac1 activity compared with tumors with low Rac1 activity (Fig. 3F). Thus, these data suggest that the PI3K/Akt pathway is upstream of Rac1 and activates Rac1, while the JNK pathway is downstream of Rac1, and that the PI3K/Akt–Rac1–JNK axis may have a significant role in the maintenance of gastric CSCs. More specific inhibition of these pathway proteins is needed to confirm these findings.

Rac1 promotes gastric adenocarcinoma CSC migration and invasion

Rac1 is known to act through a variety of effectors to control actin-myosin–dependent cell contractility and cellular motility (28, 29). We examined the role of Rac1 in the migration and invasion of gastric CSCs. AGS, MKN-45, and NCI-N87 cells grown as spheroids had increased expression of N-cadherin, which is a cell surface marker of EMT, and increases in Slug, which is transcription factor associated with EMT (Fig. 4A). Levels of two other EMT transcription factors, Snail and Zeb1, were variably changed in spheroid cells compared with monolayer cells. Spheroid cells transduced with Rac1 shRNA had reduced protein expression of N-cadherin and Slug as measured by Western blot analysis and immunofluorescence (Fig. 4B and C). Gastric cancer cells when grown as spheroid had 4.4- to 6.3-fold greater migration and 4.2- to 12.4-fold greater invasion compared with monolayer cells (Supplementary Fig. S4A). Rac1 shRNA drastically diminished the migration and invasion capabilities of gastric spheroid cells (Fig. 4D and E).

Figure 4.

Rac1 activity promotes EMT, migration, and invasion in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of N-cadherin, Slug, Snail, and Zeb1 in gastric adenocarcinoma monolayer and spheroid cells. B, Western blot analysis demonstrating levels of N-cadherin, Slug, Snail, and Zeb1 in gastric adenocarcinoma spheroid cells transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). C, Immunofluorescence photos of AGS spheroids transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). Photos and graphs of migration (D) and invasion (E) assays for gastric adenocarcinoma spheroid cells transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). Bars, SD. *, P < 0.05 compared with control.

Figure 4.

Rac1 activity promotes EMT, migration, and invasion in gastric adenocarcinoma CSCs. A, Western blot analysis demonstrating levels of N-cadherin, Slug, Snail, and Zeb1 in gastric adenocarcinoma monolayer and spheroid cells. B, Western blot analysis demonstrating levels of N-cadherin, Slug, Snail, and Zeb1 in gastric adenocarcinoma spheroid cells transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). C, Immunofluorescence photos of AGS spheroids transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). Photos and graphs of migration (D) and invasion (E) assays for gastric adenocarcinoma spheroid cells transduced with Rac1 shRNA (sh.Rac1) or scrambled shRNA (sh.Scr). Bars, SD. *, P < 0.05 compared with control.

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Treatment of gastric cancer spheroids cells with the PI3K inhibitor LY294002 significantly reduced levels of N-cadherin and Slug (Supplementary Fig. S4B). When LY294002 was added to spheroid cells in migration and invasion assays, the PI3K inhibitor blocked migration by 83%–85% and blocked invasion by 83%–88% (Supplementary Fig. S4C and S4D). The JNK inhibitor SP600125 also significantly reduced levels of N-cadherin and Slug in gastric cancer spheroid cells (Supplementary Fig. S5A). When SP600125 was added to spheroid cells in migration and invasion assays, the JNK inhibitor blocked migration by 75%–82% and blocked invasion by 79%–85% (Supplementary Fig. S5B and S5C). These data demonstrate that the PI3K/Akt–Rac1–JNK axis promotes gastric adenocarcinoma CSC EMT, migration, and invasion.

Rac1 inhibition reverses chemotherapy resistance in gastric adenocarcinoma CSCs

Numerous studies have demonstrated that CSCs are generally resistant to chemotherapy (30). We examined the sensitivity of monolayer and spheroid gastric cancer cells to two commonly used gastric adenocarcinoma chemotherapies, 5-fluorouracil, and cisplatin (31). For monolayer cells, the lethal dose required to kill 50% of cells (LD50) was 2.6–4.8 μmol/L for monolayers cells and 11.8–18.5 μmol/L for spheroid cells (Fig. 5A). Rac1 shRNA reduced proliferation in monolayer and spheroid cells by 11.7%–21.5% and 15.2%–25.1%, respectively (Fig. 5B and C). The combination of Rac1 shRNA and chemotherapy had little or no additive effect over chemotherapy alone on monolayer cells (Fig. 5B). However, there was a highly synergistic effect on spheroid cells when Rac1 inhibition was combined with chemotherapy to spheroid cells, with decreases in cell viability ranging from 62% to 66.8% (Fig. 5C). We measured apoptosis by Western blotting for cleaved caspase-3 and found that Rac1 shRNA combined with 5-fluorouracil or cisplatin led to significant induction of apoptosis in AGS spheroid cells (Fig. 5D). When the Rac1 inhibitor NSC23766 was used instead of Rac1shRNA in the above experiments, we obtained nearly identical results (Supplementary Fig. S6A–S6C). In addition, similar inhibition of spheroid cell proliferation was obtained when the PI3K inhibitor LY294002 or the JNK inhibitor SP600125 was combined with 5-fluorouracil or cisplatin (Supplementary Fig. S6D and S6E).

Figure 5.

Rac1 activity promotes chemoresistance in gastric adenocarcinoma CSCs. A, Lethal dose 50 (LD50) of gastric adenocarcinoma monolayer and spheroid cells for 5-fluorouracil (5-FU) and cisplatin. Proliferation assays for gastric adenocarcinoma monolayer cells (B) and spheroid cells (C) following treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), 5-fluorouracil (5-FU) or cisplatin chemotherapy, and/or carrier (DMSO). D, Western blot analysis of AGS spheroid cells for total Rac1 and cleaved caspase-3 following treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), 5-fluorouracil (5-FU), or cisplatin chemotherapy, and/or carrier (DMSO). Bars represent SD. *, P < 0.05.

Figure 5.

Rac1 activity promotes chemoresistance in gastric adenocarcinoma CSCs. A, Lethal dose 50 (LD50) of gastric adenocarcinoma monolayer and spheroid cells for 5-fluorouracil (5-FU) and cisplatin. Proliferation assays for gastric adenocarcinoma monolayer cells (B) and spheroid cells (C) following treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), 5-fluorouracil (5-FU) or cisplatin chemotherapy, and/or carrier (DMSO). D, Western blot analysis of AGS spheroid cells for total Rac1 and cleaved caspase-3 following treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), 5-fluorouracil (5-FU), or cisplatin chemotherapy, and/or carrier (DMSO). Bars represent SD. *, P < 0.05.

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The effects of Rac1 pathway inhibition and cisplatin chemotherapy were next examined on MKN-45 mouse xenografts. Stable transduction of Rac1 shRNA or scrambled control shRNA into MKN-45 cells did not alter cell proliferation in vitro (Supplementary Fig. S7A, S7B). MKN-45 cells stably transduced with Rac1 shRNA or scrambled control shRNA were injected into the flanks of immunodeficient mice. Once tumors reach 50–100 mm3, mice were randomized to treatment with cisplatin or PBS. Control tumors treated with PBS alone grew to over 800–900 mm3 in just 15 days following randomization (Fig. 6A). Tumors transduced with Rac1 shRNA or treated with cisplatin grew to an average size of 549 mm3 and 420 mm3, respectively. The combination of Rac1 shRNA and cisplatin dramatically inhibited tumor growth, with tumors growing to an average of 195 mm3 (76.9% less of control tumors) during the treatment period. After 15 days, xenografts were harvested and analyzed. The combination of Rac1 shRNA and cisplatin resulted in dramatic decreases in the CSC marker CD44 and the self-renewal transcription factor Sox2 (but not in Oct-4, Nanog, and c-Myc) as measured immunofluorescence (Fig. 6B; Supplementary Fig. S7C), Western blot analysis (Supplementary Fig. S7D), and IHC (Supplementary Fig. S7E). The combination of Rac1 shRNA and cisplatin had minor effects on cell proliferation as measured by PCNA (Fig. 6C) but did cause more than additive increases in overall apoptosis as measured by cleaved caspase-3 expression (Fig. 6D). Thus gastric CSCs are relatively resistant to chemotherapy, and this resistance can be overcome with Rac1 pathway inhibition.

Figure 6.

A, Tumor growth curves for MKN-45 xenografts treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), cisplatin, and/or PBS. B, Photos following IHC analysis of treated tumors for CD44 (green), Sox2 (red), Oct-4 (white), Nanog (yellow), and c-Myc (tomato). Scale bar, 20 μm. Photos and graphs of IHC analysis of treated tumors for proliferation (PCNA; C) and apoptosis (cleaved caspase-3; D). Scale bar 20 μm. E, Kaplan–Meier overall survival curves for patients undergoing surgical resection for gastric adenocarcinoma stratified by low versus high Rac1 activity in the primary tumor. F, Diagram of Rac1 pathway in gastric adenocarcinoma CSCs. Bars represent standard deviation. *, P < 0.05 compared with control.

Figure 6.

A, Tumor growth curves for MKN-45 xenografts treatment with Rac1 shRNA (sh.Rac1), scrambled control shRNA (sh.Scr), cisplatin, and/or PBS. B, Photos following IHC analysis of treated tumors for CD44 (green), Sox2 (red), Oct-4 (white), Nanog (yellow), and c-Myc (tomato). Scale bar, 20 μm. Photos and graphs of IHC analysis of treated tumors for proliferation (PCNA; C) and apoptosis (cleaved caspase-3; D). Scale bar 20 μm. E, Kaplan–Meier overall survival curves for patients undergoing surgical resection for gastric adenocarcinoma stratified by low versus high Rac1 activity in the primary tumor. F, Diagram of Rac1 pathway in gastric adenocarcinoma CSCs. Bars represent standard deviation. *, P < 0.05 compared with control.

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To determine whether Rac1 activity in human gastric adenocarcinoma tumors correlated with outcomes, we examined 47 tumor samples from gastric adenocarcinoma patients who underwent surgical resection of their gastric tumors. Given there is no reliable antibody to detect active Rac1 in human tissues, we used an antibody for phosphorylated Rac1. Phosphorylation of Rac1 is associated with decreased Rac1 activity (32, 33). Thus, phosphorylated Rac1 is inversely associated with Rac1 activity. Clinicopathologic information of these patients is given in Supplementary Table S1. Patients with tumors having high Rac1 activity had significantly worse overall survival than patients with tumors having low Rac1 activity (Fig. 6E). Figure 6F gives a schematic of PI3K/Akt–Rac1–JNK axis signaling in gastric CSCs.

This study demonstrates the vital role of the Rac1 in maintenance of gastric adenocarcinoma CSCs along with the CSC features of EMT and chemotherapy resistance. Gastric adenocarcinoma cell lines grown as spheroids had enrichment of the gastric adenocarcinoma CSC marker CD44 as well as high levels of Rac1 activity. Inhibition of Rac1 in gastric adenocarcinoma CSCs using shRNA knockdown or pharmacologic inhibition diminished expression of the stem cell transcription factor Sox-2, blocked spheroid colon formation in vitro, and blocked tumor formation in immunodeficient mice. Gastric adenocarcinoma CSCs were resistant in vitro to 5-fluorouracil or cisplatin chemotherapy, and this chemotherapy resistance could be reversed with Rac1 pathway inhibition. Rac1 inhibition in gastric adenocarcinoma xenografts acted in concert with chemotherapy to block tumor growth, and histologic examination of treated tumors showed dramatic increases in tumor cell apoptosis and depletion of CD44+ cells. Finally, examination of tumor specimens from patients with gastric adenocarcinoma who underwent surgical resection of their gastric tumors revealed an association between high Rac1 activity in their tumors and worse overall survival. These results collectively support the idea that inhibition of the Rac1 pathway may provide a novel therapeutic target for combating metastasis and chemoresistance in patients with gastric adenocarcinoma.

The EMT program is a naturally occurring transdifferentiation program that regulates changes in cell states between epithelial and mesenchymal (34). This process is vital for cancer cells to proliferate and metastasize. The link between EMT and CSCs has been examined in numerous studies, but the recently uncovered link between the passage through EMT and the acquisition of stem-like properties suggests that EMT may be a mechanism for generating CSCs (35). In this study, we use spheroid formation and expression of CD44 as means of identifying gastric adenocarcinoma CSCs. These spheroid cells or CD44+ cells have elevated expression of the EMT-associated proteins Slug and N-cadherin and significantly increased migration and invasion. Blockade of the Rac1 activity in gastric adenocarcinoma CSCs downregulates Slug and N-cadherin and reduces migration and invasion. Thus, we find in this study that targeting the Rac1 pathway affects both CSC and EMT-associated phenotypes.

Multidrug chemotherapy with agents such as 5-fluorouracil, oxaliplatin, epirubicin, and docetaxel is standard for advanced or metastatic gastric adenocarcinoma (3). Targeted therapies either alone or in combination with chemotherapy have demonstrated improved efficacy in a variety of solid tumors including gastric adenocarcinoma. Deng and colleagues performed a comprehensive survey of genomic alterations in gastric adenocarcinoma and found the existence of five distinct subgroups defined by signature genomic alterations in fibroblast growth factor receptor 2 (FGFR2), V-Ki-ras Kirsten rat sarcoma viral oncogene homolog (KRAS), EGFR, HER2, and c-MET (36). In addition to these five pathways for gastric cancer tumor growth, the VEGF-A pathway plays an important role in driving tumor angiogenesis in gastric adenocarcinoma (37). It remains unclear whether gastric adenocarcinoma CSCs may also be resistant to targeted therapies. A small percentage of gastric adenocarcinoma overexpress HER-2, and the addition of trastuzumab to chemotherapy prolonged survival in these patients from 11 to 14 months in a randomized trial (38). However, combining cytotoxic chemotherapy with agents targeting the VEGF-A or EGF pathways have not been demonstrated to increase survival in unselected gastric cancer patients (39–41). Clearly, targeted agents must be matched with the correct patient population, and it is quite possible that Rac1 pathway inhibition may only be effective for just a subset of gastric adenocarcinomas with high Rac1 activity.

Rho GTPases including Rac1 are considered undruggable because of structure–function considerations (16). While this may not be the case in the future, alternative methods for blocking Rac1 activity in gastric adenocarcinoma CSCs are currently needed. In this study, we investigated the PI3K/Akt and MAPK pathways given these pathways play a key role in CSC biology (25–27). In gastric adenocarcinoma CSCs, the PI3K/Akt pathway was found to be upstream of Rac1, and JNK was found to be downstream. Inhibition of any component of the PI3K/Akt–Rac1–JNK axis in gastric adenocarcinoma cells blocked expression of the EMT transcription factor Slug along with the EMT phenotypes of migration and invasion. Furthermore, inhibition of PI3K/Akt or JNK reversed chemotherapy resistance in gastric adenocarcinoma CSCs to a similar in magnitude as Rac1 inhibition. Thus, targeting the PI3K/Akt–Rac1–JNK pathway in gastric adenocarcinoma CSCs with available drugs for PI3K/Akt and JNK is currently possible. A significant limitation of this study is that we used nonspecific inhibitors of these pathway proteins, and thus these findings need confirmation using more specific inhibition.

In conclusion, we define the role of the Rac1 pathway in gastric adenocarcinoma, and specifically find that that Rac1 activity plays a major role in maintaining gastric adenocarcinoma CSC phenotypes. We see from our correlative studies in patient tumor samples that Rac1 is a marker of poor overall survival, and targeting this pathway is a promising new strategy.

No potential conflicts of interest were disclosed.

Conception and design: C. Yoon, S.S. Yoon

Development of methodology: C. Yoon, S.S. Yoon

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Yoon, K.K. Chang, D.J. Park, S.S. Yoon

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Yoon, S.-J. Cho, K.K. Chang, S. Ryeom, S.S. Yoon

Writing, review, and/or revision of the manuscript: S.-J. Cho, K.K. Chang, D.J. Park, S. Ryeom, S.S. Yoon

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Yoon, S. Ryeom, S.S. Yoon

Study supervision: S.S. Yoon

This study was supported from a grant from the Korean Society of Gastroenterology (to S.J. Cho), NIH/NCI Cancer Center Support Grant P30 CA008748 (to S.S. Yoon), and a MSKCC Functional Genomics Initiative Rapid Response Pilot Grant (to S.S. Yoon).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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