PI3K/Akt pathway and Nanog maintain cancer stem cells in sarcomas.

The self-renewal transcription factor Nanog and the phosphoinositide 3-kinase (PI3K)-Akt pathway are known to be essential for maintenance of mesenchymal stem cells. We evaluated their contribution to the maintenance of CD133(+) cancer stem-like cells (CSCs) and spheroid-forming cells in patient-derived cell lines from three human sarcoma subtypes: HT1080 fibrosarcoma, SK-LMS-1 leiomyosarcoma, and DDLS8817 dedifferentiated liposarcoma. Levels of Nanog and activated Akt were significantly higher in sarcoma cells grown as spheroids or sorted for CD133 expression to enrich for CSCs. shRNA knockdown of Nanog decreased spheroid formation 10- to 14-fold, and reversed resistance to both doxorubicin and radiation in vitro and in H1080 flank xenografts. In the HT1080 xenograft model, doxorubicin and Nanog knockdown reduced tumor growth by 34% and 45%, respectively, and the combination reduced tumor growth by 74%. Using a human phospho-kinase antibody array, Akt1/2 signaling, known to regulate Nanog, was found to be highly activated in sarcoma spheroid cells compared with monolayer cells. Pharmacologic inhibition of Akt using LY294002 and Akt1/2 knockdown using shRNA in sarcoma CSCs decreased Nanog expression and spheroid formation and reversed chemotherapy resistance. Akt1/2 inhibition combined with doxorubicin treatment of HT1080 flank xenografts reduced tumor growth by 73%. Finally, in a human sarcoma tumor microarray, expression of CD133, Nanog, and phospho-Akt were 1.8- to 6.8-fold higher in tumor tissue compared with normal tissue. Together, these results indicate that the Akt1/2-Nanog pathway is critical for maintenance of sarcoma CSCs and spheroid-forming cells, supporting further exploration of this pathway as a therapeutic target in sarcoma.

KRAS Activation in Gastric Adenocarcinoma Stimulates Epithelial-to-Mesenchymal Transition to Cancer Stem-Like Cells and Promotes Metastasis.

Our previous work showed that in a mouse model of gastric adenocarcinoma with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a. Tcon mice) accelerates tumorigenesis and metastasis. Here, we sought to examine KRAS activation in epithelial-to-mesenchymal transition (EMT) and generation of cancer stem-like cells (CSC). Transduction of nontransformed HFE-145 gastric epithelial cells with oncogenic KRASG12V significantly decreased expression of the epithelial marker E-cadherin, increased expression of the mesenchymal marker vimentin and the EMT transcription factor Slug, and increased migration and invasion by 15- to 17-fold. KRASG12V also increased expression of self-renewal proteins such as Sox2 and increased spheroid formation by 2.6-fold. In tumor-derived organoids from Tcon mice, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown or MEK inhibition in patient tumor-derived gastric adenocarcinoma cell lines (AGS and KATOIII). KRAS inhibition in gastric adenocarcinoma spheroid cells led to reduced AGS flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. In a tissue microarray of human gastric adenocarcinomas from 115 patients, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival. In conclusion, KRAS activation in gastric adenocarcinoma cells stimulates EMT and transition to CSCs, thus promoting metastasis. IMPLICATIONS: This study provides rationale for examining inhibitors of KRAS to block metastasis and reverse chemotherapy resistance in gastric adenocarcinoma patients.

KMT2C Mutations in Diffuse-Type Gastric Adenocarcinoma Promote Epithelial-to-Mesenchymal Transition.

PURPOSE: Lauren diffuse-type gastric adenocarcinomas (DGAs) are generally genomically stable. We identified lysine (K)-specific methyltransferase 2C (KMT2C) as a frequently mutated gene and examined its role in DGA progression. EXPERIMENTAL DESIGN: We performed whole exome sequencing on tumor samples of 27 patients with DGA who underwent gastrectomy. Lysine (K)-specific methyltransferase 2C (KMT2C) was analyzed in DGA cell lines and in patient tumors. RESULTS: KMT2C was the most frequently mutated gene (11 of 27 tumors [41%]). KMT2C expression by immunohistochemistry in tumors from 135 patients with DGA undergoing gastrectomy inversely correlated with more advanced tumor stage (P = 0.023) and worse overall survival (P = 0.017). KMT2C shRNA knockdown in non-transformed HFE-145 gastric epithelial cells promoted epithelial-to-mesenchymal transition (EMT) as demonstrated by increased expression of EMT-related proteins N-cadherin and Slug. Migration and invasion in gastric epithelial cells following KMT2C knockdown increased by 47- to 88-fold. In the DGA cell lines MKN-45 and SNU-668, which have lost KMT2C expression, KMT2C re-expression decreased expression of EMT-related proteins, reduced cell migration by 52% to 60%, and reduced cell invasion by 50% to 74%. Flank xenografts derived from KMT2C-expressing DGA organoids, compared with wild-type organoids, grew more slowly and lost their infiltrative leading edge. EMT can lead to the acquisition of cancer stem cell (CSC) phenotypes. KMT2C re-expression in DGA cell lines reduced spheroid formation by 77% to 78% and reversed CSC resistance to chemotherapy via promotion of DNA damage and apoptosis. CONCLUSIONS: KMT2C is frequently mutated in certain populations with DGA. KMT2C loss in DGA promotes EMT and is associated with worse overall survival.

Platelet-derived growth factor receptor-α and -ß promote cancer stem cell phenotypes in sarcomas.

Sarcomas are malignant tumors derived from mesenchymal tissues and may harbor a subset of cells with cancer stem-like cell (CSC) properties. Platelet-derived growth factor receptors α and ß (PDGFR-α/ß) play an important role in the maintenance of mesenchymal stem cells. Here we examine the role of PDGFR-α/ß in sarcoma CSCs. PDGFR-α/ß activity and the effects of PDGFR-α/ß inhibition were examined in 3 human sarcoma cell lines using in vitro assays and mouse xenograft models. In all three cell lines, PDGFR-α/ß activity was significantly higher in cells grown as spheroids (to enrich for CSCs) and in cells sorted for CD133 expression (a marker of sarcoma CSCs). Self-renewal transcription factors Nanog, Oct4, and Slug and epithelial-to-mesenchymal transition (EMT) proteins Snail, Slug, and Zeb1 were also significantly higher in spheroids cells and CD133(+) cells. Spheroid cells and CD133(+) cells demonstrated 2.9- to 4.2-fold greater migration and invasion and resistance to doxorubicin chemotherapy. Inhibition of PDGFR-α/ß in CSCs using shRNA or pharmacologic inhibitors reduced expression of certain self-renewal and EMT proteins, reduced spheroid formation by 74-82%, reduced migration and invasion by 73-80%, and reversed chemotherapy resistance. In mouse xenograft models, combining PDGFR-α/ß inhibition (using shRNA or imatinib) with doxorubicin had a more-than-additive effect in blocking tumor growth, with enhanced apoptosis, especially in CD133(+) cells. These results indicate that PDGFR-α/ß activity is upregulated in sarcoma CSCs and promote CSC phenotypes including migration, invasion, and chemotherapy resistance. Thus, the PDGFR-α/ß pathway represents a new potential therapeutic target to reduce metastatic potential and increase chemosensitivity.

Lauren Histologic Type Is the Most Important Factor Associated With Pattern of Recurrence Following Resection of Gastric Adenocarcinoma.

OBJECTIVE: To examine sites of initial recurrence in patients after resection of gastric and gastroesophageal junction Siewert II/III adenocarcinoma (GA). BACKGROUND: There are few recent studies on recurrence for Western patients following potentially curative resection of GA. METHODS: A review of a prospectively maintained, single institution database was performed. Clinicopathologic factors, site(s) of initial recurrence, disease-free survival, and overall survival were examined. RESULTS: From January 2000 to June 2010, 957 patients underwent potentially curative resection for GA, 435 patients (46%) had recurrent disease, and complete data on recurrence site(s) could be obtained in 386 patients. Tumors were Lauren intestinal type in 206 (53%) and diffuse or mixed-type in 180 (47%). Median time to recurrence was 12 months and 75% of recurrences occurred within 2 years. There was a significant difference in pattern of initial recurrence between the intestinal and diffuse/mixed cohorts (P < 0.001). For intestinal tumors, distant metastasis was the most common site (54%), followed by locoregional (20%), peritoneal (15%), and multifocal (11%). For diffuse/mixed tumors, peritoneal recurrence was the most common (37%), followed by distant (32%), locoregional (22%), and multifocal (9%). On multivariate analysis, Lauren histologic type was the only significant factor that was associated with both peritoneal recurrence (diffuse, hazard ratio 2.22, confidence interval 1.38-3.94) and distant recurrence (intestinal, hazard ratio 1.888, confidence interval 1.202-2.966). After recurrence, median overall survival was only 8.4 months. CONCLUSIONS: In GA patients who recur after resection, patterns of recurrence vary significantly based on Lauren histologic type.

Role of Rac1 Pathway in Epithelial-to-Mesenchymal Transition and Cancer Stem-like Cell Phenotypes in Gastric Adenocarcinoma.

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.

Linear-Stapled Side-to-Side Esophagojejunostomy with Hand-Sewn Closure of the Common Enterotomy After Prophylactic and Therapeutic Total Gastrectomy.

After total gastrectomy, anastomosis-related complications such as leak or stricture can be highly morbid. Between July 2005 and December 2015, a linear-stapled side-to-side esophagojejunostomy with hand-sewn closure of the common enterotomy (modified Orringer technique) was used for Roux-en-Y reconstruction after prophylactic total gastrectomy in 22 germline CDH1 mutation carriers and after therapeutic total gastrectomy in 18 patients diagnosed with gastric adenocarcinoma. All operations were performed by the same surgeon. No patient in either cohort developed a clinically evident anastomotic leak, one patient (2.5%) developed a contained radiographic leak that healed without intervention, and one patient (2.5%) developed an anastomotic stricture treated by endoscopic dilatation 7 months after operation. These rates were lower than radiographic leak and stricture rates in a comparison group of 32 patients who received a completely hand-sewn esophagojejunostomy (6.3 and 3.1%, respectively). Here, we describe how to perform the linear-stapled esophagojejunostomy anastomosis.

Increased RhoA Activity Predicts Worse Overall Survival in Patients Undergoing Surgical Resection for Lauren Diffuse-Type Gastric Adenocarcinoma.

BACKGROUND: Several studies have reported a high rate of RHOA mutations in the Lauren diffuse-type gastric adenocarcinoma (GA) but not in intestinal-type GA. The aim of this study was to determine if RhoA activity is prognostic for overall survival (OS) in patients with resectable GA. METHODS: Retrospective review was performed on a prospective database of GA patients who underwent potentially curative resection between 2003 and 2012 at a single institution. Tissue microarrays were constructed from surgical specimens and analyzed for phosphorylated RhoA, a marker of inactive RhoA signaling. OS was estimated by the Kaplan-Meier method, and multivariate analysis was performed by Cox proportional hazards regression modeling. RESULTS: One hundred thirty-six patients with diffuse-type GA and 129 patients with intestinal-type GA were examined. Compared to intestinal-type GA, diffuse-type GA tumors were significantly associated with increased tumor size and advanced tumor, node, metastasis (TNM) classification system stage. In patients with diffuse-type GA, high RhoA activity was associated with significantly worse OS when compared to low RhoA activity (5-year OS 52.5 vs. 81.0 %, p = 0.017). This difference in OS was not observed in patients with intestinal-type GA (5-year OS 83.9 vs. 81.6 %, p = 0.766). On multivariate analysis of diffuse-type GA patients, high RhoA activity was an independent negative prognostic factor for OS (hazard ratio 2.38, 95 % confidence interval 1.07-5.28). CONCLUSIONS: Increased RhoA activity is predictive of worse OS in patients with diffuse-type GA who undergo potentially curative surgical resection. Along with findings from genomic studies, these results suggest RhoA may be a novel therapeutic target in diffuse-type GA.

Multimodal targeting of tumor vasculature and cancer stem-like cells in sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy.

Vascular endothelial growth factor A (VEGF-A) inhibition with pazopanib is an approved therapy for sarcomas, but likely results in compensatory pathways such as upregulation of hypoxia inducible factor 1α (HIF-1α). In addition, cancer stem-like cells can preferentially reside in hypoxic regions of tumors and be resistant to standard chemotherapies. In this study, we hypothesized that the combination of VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy with evofosfamide would be an effective multimodal strategy. Multimodal therapy was examined in one genetically engineered and two xenograft mouse models of sarcoma. In all three models, multimodal therapy showed greater efficacy than any single agent therapy or bimodality therapy in blocking tumor growth. Even after cessation of therapy, tumors treated with multimodal therapy remained relatively dormant for up to 2 months. Compared to the next best bimodality therapy, multimodal therapy caused 2.8-3.3 fold more DNA damage, 1.5-2.7 fold more overall apoptosis, and 2.3-3.6 fold more endothelial cell-specific apoptosis. Multimodal therapy also decreased microvessel density and HIF-1α activity by 85-90% and 79-89%, respectively, compared to controls. Sarcomas treated with multimodal therapy had 95-96% depletion of CD133(+) cancer stem-like ells compared to control tumors. Sarcoma cells grown as spheroids to enrich for CD133(+) cancer stem-like cells were more sensitive than monolayer cells to multimodal therapy in terms of DNA damage and apoptosis, especially under hypoxic conditions. Thus multimodal therapy of sarcomas with VEGF-A inhibition, HIF-1α inhibition, and hypoxia-activated chemotherapy effectively blocks sarcoma growth through inhibition of tumor vasculature and cancer stem-like cells.

Comparison of Perioperative Radiation Therapy and Surgery Versus Surgery Alone in 204 Patients With Primary Retroperitoneal Sarcoma: A Retrospective 2-Institution Study.

OBJECTIVE: To compare outcomes of patients with retroperitoneal or pelvic sarcoma treated with perioperative radiation therapy (RT) versus those treated without perioperative RT. BACKGROUND: RT for retroperitoneal or pelvic sarcoma is controversial, and few studies have compared outcomes with and without RT. METHODS: Prospectively maintained databases were reviewed to retrospectively compare patients with primary retroperitoneal or pelvic sarcoma treated during 2003-2011. Multivariate Cox regression models were used to assess associations with the primary endpoints: local recurrence-free survival (LRFS) and disease-specific survival. RESULTS: At 1 institution, 172 patients were treated with surgery alone, whereas at another institution 32 patients were treated with surgery and perioperative proton beam RT or intensity-modulated RT with or without intraoperative RT. The groups were similar in age, tumor size, grade, and margin status (all P > 0.08). The RT group had a higher percentage of pelvic tumors (P = 0.03) and a different distribution of histologies (P = 0.04). Perioperative morbidity was higher in the RT group (44% vs 16% of patients; P = 0.004). After a median follow-up of 39 months, 5-year LRFS was 91% (95% confidence interval, 79%-100%) in the RT group and 65% (57%-74%) in the surgery-only group (P = 0.02). On multivariate analysis, RT was associated with better LRFS (hazard ratio, 0.26; P = 0.03). Five-year disease-specific survival was 93% (95% confidence interval, 82%-100%) in the RT group and 85% (78%-92%) in the surgery-only group (P = 0.3). CONCLUSIONS: The addition of advanced-modality RT to surgery for primary retroperitoneal or pelvic sarcoma was associated with improved LRFS, although this did not translate into significantly better disease-specific survival. This treatment strategy warrants further investigation in a randomized trial.

D2 lymphadenectomy with surgical ex vivo dissection into node stations for gastric adenocarcinoma can be performed safely in Western patients and ensures optimal staging.

BACKGROUND: The AJCC recommends examination of >16 nodes to stage gastric adenocarcinoma. D2 lymphadenectomy (LAD) followed by surgical ex vivo dissection (SEVD) into nodal stations is standard at many high-volume Asian centers, but potential increases in morbidity and mortality have slowed adoption of D2 LAD in some Western centers. METHODS: A total of 331 patients with gastric adenocarcinoma who underwent surgical resection at one Western institution from 1995 to 2010 were examined. RESULTS: Median age of patients was 69 years old, 65% were male, and 84% were white. D1 LAD was performed in 285 patients (86%) and D2 LAD in 46 patients (14%), with SEVD being performed in 17 patients (37%) in the D2 group. D2 LAD with or without SEVD was performed much more commonly between 2006 and 2010. For the D1, D2 without SEVD, and D2 with SEVD groups, the median number of examined nodes and percentage with >16 examined nodes were 16 and 51%, 27 and 93%, and 40 and 100%, respectively. Major complications occurred in 16% of the D1 group and 17% of the D2 group (p>0.05), and 30-day mortality was 3% for the D1 group and 0% for the D2 group. D2 LAD was a positive prognostic factor for overall survival on univariate (p=0.027) and multivariate analyses (p=0.005), but there were several possible confounding variables. CONCLUSIONS: D2 LAD at our Western institution was performed with low morbidity and no mortality. Optimal staging occurred after D2 LAD combined with SEVD, where a median of 40 nodes were examined and all patients had >16 examined nodes.