Do Joong Park

Combining PARP-1 Inhibition and Radiation in Ewing Sarcoma Results in Lethal DNA Damage

Ewing sarcomas (ES) harbor a chromosomal translocation that fuses the EWS gene to an ETS transcription factor, most commonly Friend leukemia integration 1 (FLI1). The EWS-FLI1 fusion protein acts in a positive feedback loop to maintain the expression of PARP-1, which is involved in repair of DNA damage. Here, we examine the effects of PARP-1 inhibition and radiation therapy on Ewing sarcomas. In proliferation assays, the Ewing sarcoma cell lines RD-ES and SK-N-MC were much more sensitive than non-Ewing sarcoma cell lines to the PARP-1 inhibitor olaparib (Ola; IC50 0.5–1 μmol/L vs. >5 μmol/L) and to radiation (IC50 2–4 Gy vs. >6 Gy). PARP-1 inhibition with short hairpin RNA (shRNA) or Ola sensitized Ewing sarcoma cells, but not non-Ewing sarcoma cells, to radiation therapy in both proliferation and colony formation assays. Using the Comet assay, radiation of Ewing sarcoma cells with Ola, compared to without Ola, resulted in more DNA damage at 1 hour (mean tail moment 36–54 vs. 26–28) and sustained DNA damage at 24 hours (24–29 vs. 6–8). This DNA damage led to a 2.9- to 4.0-fold increase in apoptosis and a 1.6- to 2.4-fold increase in cell death. The effect of PARP-1 inhibition and radiation therapy on Ewing sarcoma cells was lost when EWS-FLI1 was silenced by shRNA. A small dose of radiation therapy (4 Gy), when combined with PARP-1 inhibition, stopped the growth of SK-N-MC flank tumors xenografts. In conclusion, PARP-1 inhibition in Ewing sarcomas amplifies the level and duration of DNA damage caused by radiation therapy, leading to synergistic increases in apoptosis and cell death in a EWS-FLI1–dependent manner. Mol Cancer Ther; 12(11); 2591–600. ©2013 AACR.

Vascular Endothelial Growth Factor A Inhibition in Gastric Cancer

Angiogenesis is a vital process in the progression and metastasis of solids tumors including gastric adenocarcinoma. Tumors induce angiogenesis by secreting proangiogenic molecules such as vascular endothelial growth factor A (VEGF-A), and VEGF-A inhibition has become a common therapeutic strategy for many cancers. Several drugs targeting the VEGF-A pathway have been approved for clinical use in selected solid tumors, and several anti-VEGF-A strategies have been examined for gastric cancer. Phase II studies suggested that bevacizumab, an anti-VEGF antibody, can increase the efficacy of chemotherapy for advanced gastric cancer, but two international phase III trials failed to show an overall survival benefit. Two more recent international phase III trials have examined ramucirumab, an antibody targeting the primary receptor for VEGF-A, as second-line therapy for advanced gastric cancer and found a survival benefit both as single agent therapy and when combined with chemotherapy. Finally, correlative science studies suggest that the VEGF-A pathway may have varying importance in gastric cancer progression depending on ethnicity or race. This article will review the preclinical and clinical studies on the role of the VEGF-A pathway inhibition in gastric cancer.

Chemotherapy resistance in diffuse type gastric adenocarcinoma is mediated by RhoA activation in cancer stem-like cells

Purpose: The Lauren diffuse type of gastric adenocarcinoma (DGA), as opposed to the intestinal type (IGA), often harbors mutations in RHOA, but little is known about the role of RhoA in DGA. Experimental Design: We examined RhoA activity and RhoA pathway inhibition in DGA cell lines and in two mouse xenograft models. RhoA activity was also assessed in patient tumor samples. Results: RhoA activity was higher in DGA compared with IGA cell lines and was further increased when grown as spheroids to enrich for cancer stem-like cells (CSCs) or when sorted using the gastric CSC marker CD44. RhoA shRNA or the RhoA inhibitor Rhosin decreased expression of the stem cell transcription factor, Sox2, and decreased spheroid formation by 78% to 81%. DGA spheroid cells had 3- to 5-fold greater migration and invasion than monolayer cells, and this activity was Rho-dependent. Diffuse GA spheroid cells were resistant in a cytotoxicity assay to 5-fluorouracil and cisplatin chemotherapy, and this resistance could be reversed with RhoA pathway inhibition. In two xenograft models, cisplatin inhibited tumor growth by 40% to 50%, RhoA inhibition by 32% to 60%, and the combination by 77% to 83%. In 288 patient tumors, increased RhoA activity correlated with worse overall survival in DGA patients (P = 0.017) but not in IGA patients (P = 0.612). Conclusions: RhoA signaling promotes CSC phenotypes in DGA cells. Increased RhoA activity is correlated with worse overall survival in DGA patients, and RhoA inhibition can reverse chemotherapy resistance in DGA CSC and in tumor xenografts. Thus, the RhoA pathway is a promising new target in DGA patients. Clin Cancer Res; 22(4); 971–83. ©2015 AACR.

Prognostic Significance of Targetable Angiogenic and Growth Factors in Patients Undergoing Resection for Gastric and Gastroesophageal Junction Cancers

BackgroundCirculating factors in patients with gastric/gastroesophageal junction (GEJ) cancers may promote tumor progression and metastasis and may be targeted for therapy.MethodsSerum levels of ligands—vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF)—from four targetable pathways were measured before surgery, and levels were correlated to clinicopathologic characteristics and overall survival (OS).ResultsIn 147 patients who underwent potentially curative resection for gastric/GEJ adenocarcinoma, VEGF-A levels were higher in patients with R1 versus R0 resection (pxa0=xa00.037). High EGF levels were associated with poorly differentiated tumors (pxa0=xa00.02). Elevated FGF2 levels were found in Lauren diffuse-type tumors (pxa0=xa00.017) and tumors with seven or more metastatic nodes (N3) (pxa0<xa00.042). Patients with advanced-staged tumors had higher HGF levels (pxa0=xa00.012). At a median follow-up of 35xa0months, 46 patients (31xa0%) had died. Increased VEGF and HGF levels were correlated with decreased OS (pxa0=xa00.009 and 0.005). An adjusted total value (ATV) of all factors was better than any single factor in stratifying patients into good and poor prognosis groups (5-year OS 84.1 vs. 53.9xa0%, pxa0=xa00.005). By multivariate analysis, serum VEGF-A and ATV were significant independent prognostic factors (along with T and N category) for OS (pxa0=xa00.028 and 0.013, respectively).ConclusionsIn patients undergoing resection for gastric and GEJ cancer, high levels of angiogenic and growth factors are associated with unfavorable tumor characteristics and poorer overall survival. Thus levels of these factors can help delineate tumor biology and stratify prognosis.

Serum VEGF-A and Tumor Vessel VEGFR-2 Levels Predict Survival in Caucasian but Not Asian Patients Undergoing Resection for Gastric Adenocarcinoma

BackgroundClinical trials of agents targeting the vascular endothelial growth factor A (VEGF-A) pathway in gastric adenocarcinoma (GA) suggest that these therapies may have varying efficacy in different races.MethodsVEGF-A in serum and/or VEGF receptor 2 (VEGFR-2) in CD31-positive tumor vessels (VEGFR-2/CD31) were measured in 118 Caucasians and 263 Asians who underwent gastric resection at two institutions and correlated with overall survival (OS). Blood was drawn before any treatment. Patients receiving neoadjuvant treatment were excluded from VEGFR-2 analysis.ResultsCompared with Asians, Caucasians were older (mean age 66–73 vs 59–62xa0years), had more proximal tumors, and had more advanced TNM stage. In the VEGF-A cohort, Caucasians had a median VEGF-A level that was 95xa0% higher than that of Asians and a much higher standard deviation (88xa0±xa06.206 vs 45xa0±xa076xa0pg/ml, pxa0<xa00.001). The 5-year OS for patients with low versus high VEGF-A levels was 72 versus 43xa0% in Caucasians (pxa0=xa00.001) and 86 versus 77xa0% in Asians (pxa0=xa00.236). In the VEGFR-2 cohort, OS was worse in Caucasians with high VEGFR-2/CD31 levels (49 vs 73xa0%, pxa0=xa00.038), while there was no significant difference in OS in Asians (80 vs 90xa0%, pxa0=xa00.119). On multivariate analyses of significant prognostic factors (excluding treatment factors and margin status), serum VEGF-A and tumor VEGFR-2/CD31 levels were independent predictors of OS only in Caucasians.ConclusionsIn patients with resectable GA, VEGF-A and VEGFR-2/CD31 levels are independent predictors of OS in Caucasians but not in Asians, suggesting varying importance of this pathway in GA progression among different races.

Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

PURPOSEnTo examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma.nnnMETHODS AND MATERIALSnHypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines.nnnRESULTSnIn both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm(3) within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm(3) for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays.nnnCONCLUSIONSnInhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

Laparoendoscopic Single-Site Bariatric Surgery: A Review of Single-Port Laparoscopic and Endoscopic Bariatric Treatments

Bariatric surgery is an established and effective treatment, not only to combat morbid obesity, but also to address associated metabolic comorbidities. At this time, the cutoff for bariatric or metabolic surgery in terms of body mass index (BMI) is decreasing, making it more feasible for certain individuals to consider minimally invasive surgical options. Innovations in the technique have led to the application of laparoendoscopic single-site surgery (LESS) in the field of bariatrics, which uses a single or no incision in the performance of weight-reducing surgery. To date, there is no consensus regarding patient selection though most candidates for single-port bariatric surgery are female. Some doctors suggest that single-port bariatric surgery may not be recommended in patients with BMI of more than 50 kg/m2, height of more than 180 cm, and xiphoid–umbilicus distance of more than 20 cm. Sleeve gastrectomy (SG) is now the most widely performed bariatric surgery worldwide and single-port SG (SPSG) is already established as a routine procedure in various institutions. Current evidence shows that SPSG is less painful and demonstrates higher rates of patient satisfaction regarding the wound. SPSG is feasible and is recommendable in patients who meet certain criteria. Furthermore, endoscopic treatment modalities such as intragastric balloons and endoluminal malabsorptive devices are being developed to bridge the gap between medical and surgical treatments. Nevertheless, there is still insufficient evidence to prove the superiority of LESS bariatric surgery over conventional laparoscopic surgery. Large, well-designed prospective analyses are needed to determine the criteria for selecting patients suitable to undergo LESS bariatric surgery and to predict the procedure’s role in the growth of bariatric surgery.

Abstract 4414: RhoA activation in diffuse type gastric adenocarcinoma promotes cancer stem cell phenotypes including chemotherapy resistance

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

Abstract 5447: 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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroduction: 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.nnMethods: 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.nnResults: 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.nnConclusion: 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.nnCitation 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

Abstract 3873: Hedgehog signaling maintains gastric cancer stem cells and promotes chemotherapy resistance: results from laboratory and clinical studies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnIntroduction: 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.nnMethods 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).nnConclusions: 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.nnCitation 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