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Supplementary Figure S1 from Activation of p38 Mitogen-Activated Protein Kinase Is Required for Death Receptor–Independent Caspase-8 Activation and Cell Death in Response to Sphingosine

Supplementary Data from Oncogenic Ras Signals through Activation of Both Phosphoinositide 3-Kinase and Rac1 to Induce c-Jun NH2-Terminal Kinase–Mediated, Caspase-Independent Cell Death

Cells avert the development of malignancy in response to deregulated oncogene expression by activating a regulated cell death pathway. However, the molecular mechanism underlying this oncogene-induced cellular death process remains unclear. Here, we show that retroviral expression of oncogenic H-ras induced cell death in a caspase-independent manner in normal cells. Inhibition of c-Jun NH2-terminal kinase (JNK) by pretreatment with SP600125 or a dominant-negative form of JNK blocked cell death. Rac1 and phosphoinositide 3-kinase (PI3K) were activated in cells overexpressing oncogenic H-ras. Inhibition of Rac1 with RacN17, a dominant-negative form of Rac1, attenuated oncogenic H-ras–induced JNK activation and subsequent cell death. Interestingly, inhibition of PI3K with LY294002 or by small interfering RNA–mediated knockdown of PI3K p85 or p110 subunits also clearly attenuated JNK activation and cell death. No cross talk was observed between Rac1 and PI3K, indicating that these pathways operate in parallel. Our findings show that JNK is necessary for oncogenic H-ras–induced, caspase-independent cell death, and that both PI3K and Rac1 activities are required for JNK activation and cell death. Determining the molecular mechanisms that mediate cell death responses to deregulated oncogenes provides a more refined understanding of cellular disposal processes in normal cells and increases our appreciation of these events as a mechanism for protecting against malignant progression. (Mol Cancer Res 2009;7(9):1534–42)

Intestinal-type gastric cancer often results from Helicobacter pylori infection through intestinal metaplasia, a transdifferentiated premalignant phenotype. Because H. pylori virulence factor CagA has been associated with aberrant expression of the transcription factor CDX1, which regulates intestinal differentiation, we explored its relationship with H. pylori infection and function during gastric carcinogenesis in normal gastric epithelial cells and gastric cancer cell lines. Infection of HFE 145 cells with CagA+ H. pylori increased expression of CDX1, as well as the epithelial-to-mesenchymal transition (EMT) markers Snail and Slug, increased invasion and migration, but those effects were not found in HFE 145 cells infected with CagA-deficient H. pylori. CDX1 overexpression increased expression of the intestinal markers Villin, sucrose isomaltase (SI), and MUC2, induced spheroid formation, and enhanced expression of the stem cell markers CD44, SOX2, Oct4, and Nanog, while CDX1 knockdown inhibited proliferation and intestinal stemness. Treatment of CDX1-expressing cells with metformin, an antidiabetic drug known to decrease the risk of gastric cancer, decreased expression of EMT and stemness markers, and reduced spheroid formation. In a murine xenograft model, combining metformin or shCDX1 with cisplatin reduced tumor growth, increased caspase-3 cleavage, and reduced expression of CD44 and MMP-9 to a greater degree than cisplatin alone. Patients with more advanced intestinal metaplasia staging exhibited higher CDX1 expression than those with earlier intestinal metaplasia staging (P = 0.039), and those with H. pylori tended to have more CDX1 expression than noninfected patients (P = 0.061). Finally, human tissue samples with higher CDX1 levels showed prominent CD44/SOX2 expression. Our findings indicate CagA+ H. pylori–induced CDX1 expression may enhance gastric cancer tumorigenesis and progression, and support therapeutic targeting of CDX1 in gastric cancer. Implications: This study shows that CDX1 contributes to the tumorigenesis and progression of gastric cancer and suggests the potential of targeting CDX1 to treat this malignancy.

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.

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.

Background: TARGIT-A trial showed that intraoperative radiotherapy (IORT) using Intrabeam® concurrent with lumpectomy within a risk-adapted approach could be considered as an option for eligible patients with early breast cancer. To introduce IORT in clinical practice for Korean breast cancer patients, safety profiles for this new technique from clinical trial with Korean are required due to ethnic (anatomical) difference. Acute toxicity is a key hurdle for patients receiving IORT, because IORT may affect wound healing and increase wound complications. We conducted a phase II study as a feasibility test of IORT at the dose of 20 Gy for the replacement of boost-EBRT in Korean women who are candidates for breast-conserving treatment. Methods: This single-arm trial aimed to investigate acute toxicity occurred within 6 months after IORT. In 1,119 patients receiving EBRT from TARGIT-A trial, the incidence of acute toxicity within 6 months is 15%. Compared with WBRT, we aim to prove a non-inferiority of IORT with 20 Gy. A sample size of 195 achieves 80% power to detect a non-inferiority proportion (P0) of 0.2300 using a one-sided binomial test for non-inferiority. These results assume that the actual proportion (P1) is 0.1500. Considering a drop-out rate of 10%, the trial would need to enroll 215 patients in total. This trial is registered with ClinicalTrials.gov, number NCT02213991. Results: From August 2014 to November 2016, 233 women with early breast cancer were screened, and 215 undergoing IORT were enrolled. In 36 women, clinically significant complications during acute period are noted. The rate of patients experiencing acute toxicity was 16.7% (95% CI, 11.8-21.7%). The actual non-inferiority margin of our trial was 21.7% under the pre-specified margin as 23.0%. In details, There are 31 with seroma collection (more than 3 times when aspiration volume is over 10cc), 2 with wound infection, and 5 with skin break down. At a median follow-up of 17.8 months, there is no local recurrence. Conclusions: Targeted intraoperative radiotherapy using Intrabeam® is a safe procedure for Korean breast cancer patients with acceptable toxicity profile in acute period. Citation Format: Lee HW, Yoon C, Ahn SG, Kim JW, Cha YJ, Lee IJ, Jeong J. A prospective phase II study of TARGeted Intraoperative radioTherapy boost plus whole breast irradiation in breast cancer patients undergoing breast-conserving treatment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-11-21.

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.

Purpose: Gastric cancers may harbor a subset of cells with cancer stem cell (CSC) properties, including chemotherapy resistance, and CD44 is a gastric CSC marker. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. Here, we examine the role of HH signaling in CD44(+) gastric cancer cells. Experimental Design: Gastric cancer cell lines, tumor xenografts, and patient tumors were examined. Results: Gastric cancer cell lines AGS, MKN-45, and NCI-N87 grown as spheroids or sorted for CD44(+) were found to have upregulation of HH pathway proteins. HH inhibition using Smoothened (Smo) shRNA or vismodegib (VIS) decreased spheroid formation and colony formation. CD44(+) cells, compared with unselected cells, were also resistant to 5-fluorouracil and cisplatin chemotherapy, and this resistance was reversed in vitro and in xenografts with Smo shRNA or VIS. CD44(+) cells also had significantly more migration, invasion, and anchorage-independent growth, and these properties could all be blocked with HH inhibition. Clinical tumor samples from a phase II trial of chemotherapy with or without VIS for advanced gastric cancer were analyzed for CD44 expression. In the chemotherapy alone group, high CD44 expression was associated with decreased survival, whereas in the chemotherapy plus VIS group, high CD44 expression was associated with improved survival. Conclusions: HH signaling maintains CSC phenotypes and malignant transformation phenotypes in CD44(+) gastric cancer cells, and HH inhibition can reverse chemotherapy resistance in CD44(+) cells. Gastric cancer is a heterogeneous disease, and the strategy of combining chemotherapy with HH inhibition may only be effective in tumors with high CD44 levels. Clin Cancer Res; 20(15); 3974–88. ©2014 AACR.

Background: Newly released guideline standardizing a negative margin after breast-conservative surgery (BCS) as “no ink on tumor” by SSO-ASTRO stressed the importance of estimation tumor extent with comprehensive breast imaging studies. To evaluate clinical value of breast magnetic resonance imaging (MRI) in patients with BCS, we compared the degree of correlation between MRI-pathology and ultrasonography (US)-pathology according to subtypes. In addition, we investigated the margin-positive rates and secondary operation rates for margin clearance. Methods: We identified patients with invasive breast cancer who had preoperative breast MRI and ultrasound between 2011 and 2016. We excluded patients having large tumor more than 5cm or multiple tumors or undergoing mastectomy. Patients were classified into 4 subtypes based on the immunohistochemistry; luminal A, luminal B/HER2, HER2, triple-negative breast cancer (TNBC). Lin's concordance correlation coefficient was used to measure the agreement between the MRI or US and tumor extent. Tumor extent was defined as pathologic tumor size including in situ carcinoma. Margin-positivity was assessed based on intraoperative frozen examination. Results: A total 516 patients with single tumor undergoing BCS were included. Means of tumor size were 1.99 ± 0.91 cm by pathologic examination, 1.91 ± 1.01 cm by MRI, and 1.76 ± 0.92 cm by US, respectively. The correlation coefficient of MRI-pathology was significantly higher than that of US-pathology (r=0.6975 vs. 0.6211, P=0.001). A superiority of MRI than US in measuring pathologic extent was only observed in TNBC (r=0.8089 vs. 0.6014, P0.999). Margin positive and re-excision rates according to the subtypes Luminal A (n=302)Luminal B (n=80)HER2 (n=27)TNBC (n=107)P valuePositive margin35 (11.6)14 (17.5)8 (29.6)19 (17.8)0.0382Re-excision14 (4.6)4 (5.0)1 (3.7)5 (4.7)>0.9999 Conclusions: Given a superiority of MRI to US in preoperative assessment, MRI-guided BCS did not reduce the margin-positive rate in TNBC. In the HER2, size correlation of MRI-pathology was very low, and the margin-positive rate was high. Collectively, our findings suggest that accuracy of MRI has limited value to reduce the margin-positive rate. Citation Format: Bae SJ, Ahn SG, Yoon C, Cha YJ, Jeong J. Accuracy of breast magnetic resonance imaging has limited value to reduce the margin-positive rate: A study in relation to the molecular subtypes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-02-11.

INTRODUCTION: The Cancer Genome Atlas found that the receptor tyrosine kinase (RTK)-Ras signaling pathway is altered in half of gastric adenocarcinomas (GAs). We added oncogenic Kras to the Atp4b-Cre;Cdh1fl/fl;Trp53fl/fl mouse model of GA and found accelerated tumorigenesis and metastasis. We also found Kras activity to be higher in gastric cancer stem-like cells (CSCs). Thus we hypothesized that Kras activty is critical for maintenance of gastric CSCs and promotes CSC phenotypes. METHODS: Human and murine GA cell lines were examined. The effect of Kras pathway inhibition was examined in GA spheroid cells and monolayer cells in various in vitro assays. Kras activity and Kras pathway inhibition was examined in CSCs, primary tumors and metastases in our GA mouse model. The combination of chemotherapy and Kras pathway inhibition was tested in human GA xenografts. RESULTS: Kras inhibition with shRNA or MEK inhibitor PD0325901 decreased the ability of GA cells to form spheroids and deceased expression of the stem cell transcription factor, Sox2. CD44(+) gastric CSCs had 65.8-75.4% higher migration, 64.2-79.5% higher invasion, and 63.1-70.2% more anchorage-independent growth compared to unselected cells. These properties could all blocked by 71.1-82.7% with Kras shRNA or PD0325901. In our GA mouse model, PD0325901 starting at 6 weeks of life increased median survival from 76 days to 95 days. Primary tumors and metastases from these mice with and without treatment with MEK inhibition are being analyzed for level of CD44 CSCs, Kras activity, and extent of metastases. Gastric CSCs were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed in human GA xenografts transducing cells with Kras shRNA. CONCLUSION: Kras is more active in gastric CSCs than non-CSCs and promotes malignant phenotypes including metastasis and chemotherapy resistance. Kras pathway inhibition can block these CSC phenotypes, and thus may prove useful in various combination therapies. Note: This abstract was not presented at the meeting. Citation Format: Changhwan Yoon, Kevin K. Chang, Jacob Till, Sandra W. Ryeom, Sam S. Yoon. Oncogenic Kras activation in gastric adenocarcinoma promotes cancer stem cell phenotypes including metastasis & chemotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2897. doi:10.1158/1538-7445.AM2017-2897

Introduction: Sarcomas often thrive in hypoxic conditions to proliferate and metastasize. To survive in such an environment, sarcomas upregulate hypoxia inducible factor-1α (HIF-1α), which enhances the transcription of over 150 genes mediating tumor metabolism, angiogenesis, and metastasis. HIF-1α may be important for the maintenance of a subset of tumor cells referred to as “sarcoma stem-like cells” or SSCs. Platelet-derived growth factor receptor (PDGFR) is important in mesenchymal biology and may be a potential target of HIF-1α. Methods: Three human sarcoma cell lines were examined: HT1080 (fibrosarcoma), SK-LMS-1 (leiomyosarcoma), and DDLS8817 (dedifferentiated liposarcoma). Cell lines were grown as spheroids on ultra-low attachment dishes to enrich for SSCs. The roles of HIF-1α and PDGFR-α/β in promoting SSC phenotypes, such as migratory/invasive capacity, were assessed via several in vitro assays under hypoxic conditions. Results: Sarcoma cell lines grown as spheroids had increased levels of HIF-1α and increased phosphorylation of PDGFR-α/β as compared to when grown as monolayers; these effects were even more pronounced when sarcoma spheroids were grown under hypoxic conditions. Hypoxic conditions also increased migratory and invasive capacity in spheroid cells by 65-73% as compared to normoxic conditions. HIF-1α knockdown using shRNA in the three cell lines decreased expression of self-renewal proteins and decreased spheroid formation by 67-80%. HIF-1α knockdown decreased expression of epithelial-mesenchymal transition (EMT) transcription factors and reduced migratory and invasive capacity by 80-87% under normoxic conditions and 85-87% under hypoxic conditions. Phosphorylation of PDGFR-α/β was decreased in sarcoma cell lines following HIF-1α knockdown under normoxic and hypoxic conditions. PDGFR-α/β inhibition with imatinib reversed chemotherapy resistance in normally resistant SSCs, and treatment of HT1080 xenografts with doxorubicin chemotherapy and imatinib reduced tumor formation by 85% as compared to control and by 77% as compared to chemotherapy alone. Conclusions: The HIF-1α/PDGFR axis supports the maintenance of SSCs, especially under hypoxic conditions, and promotes SSC phenotypes such as migratory/invasive capacity and chemotherapy resistance. Combining chemotherapy with PDGFR-α/β inhibition may be a promising strategy to reverse chemotherapy resistance and prevent metastasis in sarcomas. Note: This abstract was not presented at the meeting. Citation Format: Kevin K. Chang, Changhwan Yoon, Sam S. Yoon. Hypoxia-inducible factor 1-α maintains sarcoma stem-like cells in hypoxic regions of tumors and promotes migration and invasion via upregulation of platelet-derived growth factor receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2017-2899

Purpose: The Lauren diffuse type of gastric adenocarcinoma (DGA) is genomically stable compared to the intestinal type, but the primary driver mutations in DGA are still being defined. Lysine (K) -specific methyltransferase 2C (KMT2C), also known as mixed lineage leukemia 3 (MLL3), is a histone methyltransferase involved in transcriptional coactivation and can be mutated in gastric cancer. The purpose of this study was to determine the frequency of KMT2C mutation in DGA and to elucidate the role of KMT2C in DGA tumorigenesis and progression. Methods: Whole exome sequencing was performed on matched tumor samples of 27 patients with DGA who underwent potentially curative surgical resection. Immunohistochemistry (IHC) was performed for KMT2C protein expression on these same tumors and on an additional 289 tumors from a separate cohort of patients with DGA. KMT2C overexpression in DGA cell lines (MKN-45 and SNU-668) using lentiviral transduction and KMT2C shRNA knockdown in normal gastric epithelial cells were examined in several in vitro assays. Results: Thirty-eight percent of the 27 sequenced DGA specimens harbored somatic mutations in KMT2C. The next most frequent mutation, CDH1, which encodes E-cadherin, was found in 28% of specimens. Patients with KMT2C mutation tended to have worse overall survival (OS) compared to patients without the mutation (p = 0.194). Additional tumor tissue for IHC analysis was only available from 11 of 27 patients. Among this group, patients with tumors expressing KMT2C protein had better OS compared to patients whose tumors did not express KMT2C (p = 0.034). In an external validation set of 289 DGA samples, KMT2C expression again correlated with better OS (p = 0.009). In the DGA cell lines, KMT2C overexpression reduced proliferation by 32-39%, soft agar colony formation by 75-77%, spheroid cell formation by 77-78%, cell migration by 52-60%, and cell invasion by 50-74%. KMT2C overexpression also increased resistance to 5-FU and cisplatin chemotherapy. KMT2C shRNA knockdown in gastric epithelial cells increased proliferation, soft agar colony formation, migration, and invasion by 1.7- to 25-fold. Conclusions: KMT2C is frequently mutated in DGA, and loss of KMT2C protein expression correlates with worse OS. Restoration of KMT2C expression in DGA cell lines reduces cancer stem-like cell and malignant phenotypes. Thus, KMT2C may be a novel therapeutic target for patients with DGA. Citation Format: Soo-Jeong Cho, Changhwan Yoon, Jun Ho Lee, Kevin K. Chang, Bulent A. Aksoy, Do Joong Park, Sam S. Yoon. Diffuse gastric adenocarcinoma often harbors KMT2C mutations resulting in malignant phenotypes and worse overall survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 893.

Introduction: The diffuse type of gastric adenocarcinoma (GA) was recently found to frequently harbor activating mutations in RHOA. RhoA is a member of the small GTPase-Ras-like proteins which are involved in cell signaling for cell migration and cell cycle. Methods: RhoA activity and inhibition was examined in diffuse GA cell lines grown as spheroids (i.e. cancer stem cell conditions) and as monolayers in various in vitro assays. RhoA knockdown combined with chemotherapy was examined in a mouse xenograft model. Results: RhoA activity was much higher in diffuse GA cell lines MKN-45 and SNU-668 compared to intestinal GA cell lines NCI-N87 and AGS. RhoA activity was further increased in diffuse GA cells when grown as spheroids rather than as monolayers. RhoA inhibition with shRNA or the RhoA inhibitor Rhosin decreased spheroid formation and deceased expression of the stem cell transcription factor, Sox2. Diffuse GA cells when grown as spheroids had significantly greater migration, invasion, and anchorage-independent growth, and these properties could all be blocked with RhoA shRNA or Rhosin. Diffuse GA spheroid cells (compared to monolayer cells) were resistant to 5-fluorouracil and cisplatin chemotherapy, and this chemotherapy resistance could be reversed with RhoA inhibition. In the MKN-45 xenograft model, cisplatin inhibited tumor growth by 50%, RhoA shRNA by 60%, and the combination by 83%. Clinical tumor samples of diffuse GA are currently being analyzed for RhoA expression and correlated with extent of disease and response to chemotherapy. Conclusions: RhoA signaling is upregulated in diffuse GA cells grown as spheroids and promotes malignant transformation phenotypes such as migration and invasion. RhoA inhibition can reverse chemotherapy resistance in GC spheroid cells and in tumor xenografts, and thus the RhoA pathway is a promising new target of therapy for diffuse GA. Citation Format: Changhwan Yoon, Soo-Jeong Cho, Bulent A. Aksoy, Do Joong Park, Sam S. Yoon. RhoA activation in diffuse type gastric adenocarcinoma promotes cancer stem cell phenotypes including chemotherapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4414. doi:10.1158/1538-7445.AM2015-4414

Introduction: In a previous clinical trial, we demonstrated that bevacizumab, an anti-vascular endothelial growth factor A (VEGF-A) antibody, combined with radiation leads to >80% tumor necrosis in about one-half of patients with soft tissue sarcomas. Sarcomas with a poor response to this therapy had upregulation of hypoxia inducible factor 1α (HIF-1α) and HIF-1α target genes. Here, we describe two trimodality approaches to this problem: (1) blockade of HIF-1α using genetic or pharmacologic inhibition and (2) destruction of hypoxic regions of tumors using the hypoxia-activated chemotherapeutic, TH-302. Methods: Trimodality therapies were used in a HT1080 fibrosarcoma xenograft model and the LSL-KrasG12D/+/Trp53fl/fl genetically engineered mouse model of sarcoma. Treated tumors were examined for effects on cancer cells and tumor vasculature. Trimodality therapies were also studied in vitro on four sarcoma cell lines and on two types of endothelial cells. Results: In both mouse models, trimodality therapy with HIF-1α inhibition using low dose doxorubicin or HIF-1α shRNA was significantly better than any bimodality therapy in blocking tumor growth, with tumors growing to only 13-18% size of controls. When hypoxic areas of tumors were targeted with TH-302, this alternative trimodality therapy was again better than any bimodality therapy in blocking sarcoma tumor growth (tumor size 9% of controls), and tumors failed to grow after stopping treatment for more than 2 months. Analysis of treated tumors demonstrated the predominant effect with both trimodality therapies was through induction of endothelial cell apoptosis (2.6-3.3 fold more than the best bimodality therapy) and destruction of tumor vasculature (86-89% less microvessel density than controls). When the effects of trimodality therapy were examined in vitro, decreases in proliferation and colony formation and induction of apoptosis from trimodality therapy were much more pronounced in tumor-derived endothelial cells than in sarcoma cell lines. Conclusion: HIF-1α inhibition or hypoxia-activated chemotherapy is effective when combined VEGF-A inhibition and radiation in controlling sarcomas by maximizing destruction of tumor vasculature and blocking the hypoxic response. Clinical trials are currently in development using both these trimodality strategies. Citation Format: Changhwan Yoon, Hae-June Lee, Do Joong Park, William D. Tap, T. S. Karin Eisinger-Mathason, David G. Kirsch, M. Celeste Simon, Sam S. Yoon. Targeting vascular endothelial growth factor A and tumor hypoxia combined with radiation eradicates sarcomas through destruction of tumor vasculature and thwarting of the hypoxic response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5447. doi:10.1158/1538-7445.AM2014-5447

Introduction: Gastric cancers may harbor a small subset of cancer stem cells (CSCs) with the exclusive ability to self-renew and differentiate into heterogenous cell types. These CSCs may also contribute to chemotherapy resistance. The Hedgehog (HH) pathway is a key developmental pathway that can be subverted by CSCs during tumorigenesis. However in a recent randomized phase II trial of chemotherapy with or without the small molecule HH inhibitor vismodegib for advanced gastric cancers, the addition of vismodegib did not increase progression-free survival (PFS) or overall survival (OS). In this study, we examine the role of HH signaling in gastric CSC maintenance and chemotherapy resistance. Methods and Results: Gastric cancer cell lines AGS, MKN-45, and NCI-N87 were grown as spheroids to enrich for CSCs. Spheroid cells were found to have upregulation of the putative gastric CSC marker CD44 along with HH pathway proteins Shh, Ptch1, Smo, and Gli1. Inhibition of the HH signaling using Smo shRNA or vismodegib decreased spheroid formation by 70.3-78.4% or 66.9-70.8%, respectively, and attentuated another CSC phenotype, single cell colony formation, by 66.9-78.4%. Transformation phenotypes such as migration, invasion, and anchorage-independent colony formation were also inhibited in gastric CSCs by 50.2-65.6%, 57.4-66.3%, and 3.8-4.6 fold, respectively. CD44(+) gastric CSCs from all 3 cell lines were resistant to 5-fluorouracil or cisplatin chemotherapy, and this resistance was reversed with the addition of Smo shRNA or vismodegib. The combination of Smo shRNA and cisplatin synergistically blocked the growth of MKN-45 xenografts, and treated tumors demonstrated a 1.8-2.6 fold increase in tumor cell apoptosis compared to tumors treated with cisplatin alone. Clinical tumor samples from the phase II vismodegib trial were analyzed for CD44 expression (as a surrogate to levels of CSCs). In the chemotherapy alone group, high CD44 expression was associated with worse PFS and OS. However in the chemotherapy with vismodegib group, high CD44 expression was associated with improved PFS and OS. For two patients in the vismodegib arm of the study who had a complete response, CD44 levels were 6.1-fold higher than the other patients in the group (p=0.001). Conclusions: HH signaling is required to maintain gastric CSC phenotypes such as spheroid formation and colony formation from single cells as well malignant transformation phenotypes such as migration, invasion, and anchorage-independent growth. Gastric CSCs are resistant to chemotherapy compared to unselected cells, and HH inhibition can reverse this resistance. Given gastric cancer is a heterogeneous disease, the strategy of combining chemotherapy with HH inhibition may only be effective in a subset of gastric cancer patients with high levels of CD44(+) gastric CSCs. Citation Format: Changhwan Yoon, Do Joong Park, Benjamin Schmidt, Nicholas J. Thomas, Hae-June Lee, Teresa S. Kim, Yelena Y. Janjigian, Deirdre J. Cohen, Sam S. Yoon. Hedgehog signaling maintains gastric cancer stem cells and promotes chemotherapy resistance: results from laboratory and clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2014-3873