Anish Ray

Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth

SummaryTranscription factor Specificity proteinxa01 (Sp1) and its downstream target survivin (inhibitor of apoptosisxa0protein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (pxa0<xa00.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.

Pleuropulmonary Blastoma: A Single-center Case Series of 6 Patients

Pleuropulmonary blastoma (PPB) is a rare malignancy of childhood which when left untreated often shows pathologic progression resulting in a more aggressive neoplasm with an increasingly poor prognosis. Because of this it is important to diagnose and initiate treatment early. However, early stage PPB can appear as a cystic lung lesion on imaging and can be easily misdiagnosed given the rarity of the malignancy. Moreover, current therapeutic guidelines for these lesions are not well established, making treatment decisions and management difficult for clinicians. DICER1 mutations are known to be present in a majority of PPBs with or without a germline mutation and may be part of a familial tumor predisposition syndrome. The clinical, pathologic, and genetic data of 6 patients are summarized here. Two patients with type I PPB and 4 patients with type II PPB underwent surgical and chemotherapeutic treatment and all are alive and without recurrence 1 to 13 years after treatment. With increasing awareness of PPB, it is important for clinicians to consider this malignant entity in the evaluation and treatment of patients presenting with a cystic lung abnormality, especially in cases with a history strongly suggestive of a DICER1 mutation.

Abstract 1532: Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells

Ewing sarcoma (ES) is one of the most frequent primary bone and soft tissue tumors that occur in pediatric and adolescent population. ES is often diagnosed after the disease has already metastasized. With available treatment options, the prognosis of metastatic ES patients is poor and the 5-year survival rate is less than 20%. Therefore, identifying precise targeted therapies to induce therapeutic response in ES patients is urgently needed. An oncogenic fusion protein and transcription factor EWS-FLI1 is associated with more than 85% of ES tumors. Mithramycin has been identified as an effective agent to target EWS-FLI1 and is currently in clinical trials. In this study, we investigated the efficacy of Mithramycin to induce the anti-proliferative activity of chemotherapeutic agents commonly used for the treatment of this malignancy using human ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mithramycin or Vincristine or Doxorubicin or Etoposide. Cell growth inhibition was evaluated at 24 and 48 h post-treatment using CellTiter Glo kit. Results showed a dose/time-dependent anti-proliferative effect for all agents. To confirm the effect of Mithramycin on selected cell lines, mRNA expression of downstream targets of EWS-FLI1 (ID2, LDB2, NROB1 and RCOR1 genes) was determined by quantitative PCR following treatment with Mithramycin. Mithramycin significantly down-regulated all tested genes and these results are in agreement with published work. In order to determine the combination response, cell growth inhibition of the chemotherapeutic drugs was assessed in the presence of added Mithramycin. The combination Index was evaluated by Chou-Talalay method. Further studies were performed for the combination of Mithramycin and Vincristine to investigate its effect on apoptosis and cell cycle arrest using CHLA10 and TC205 cells. Apoptotic markers such as the expression of cleaved Poly (ADP-ribose) polymerase (c-PARP) and survivin were determined by Western blot analysis. The apoptotic cell population was measured by Annexin-V staining using flow cytometry. When compared to individual agents, the combination of Mithramycin and Vincristine showed greater effect on apoptosis in both cell lines as evinced by an increase in Annexin-V positive cells, decrease in survivin expression and up-regulation of c-PARP. Overall, these results indicate the potential clinical advantage of combination treatment involving Mithramycin along with other chemotherapeutic agents for inhibiting the growth of ES cells. Molecular profiling analysis is underway to elucidate the candidate pathways associated with beneficial effect of this combination treatment. Note: This abstract was not presented at the meeting. Citation Format: Anish Ray, Bhavani Nagarajan, Umesh T. Sankpal, Sagar Shelake, W. Paul Bowman, Andras Lacko, Riyaz M. Basha. Mithramycin induces the antiproliferative activity of chemotherapeutic agents in Ewing sarcoma cells [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 1532. doi:10.1158/1538-7445.AM2017-1532

Abstract 2197: Effect of tolfenamic acid on the therapeutic efficacy of chemotherapeutic drugs used in the treatment of Ewing sarcoma

Ewing sarcoma (ES) is the second most common pediatric primary bone tumor, and affects children between the ages of 1 to 20 years. With the advancement in therapeutic options the 5-year event free survival rate of patients with localized tumor has gone up to 70%. However, in patients with metastases at diagnosis, this rate is less than 20%. One of the distinguishing hallmarks of ES is the presence of oncogenic fusion transcription factor formed by the translocation between EWS and ETS family transcription factor. The EWS-FLI1 translocation is most commonly found in ES patients. Therefore targeting EWS-FLI1 transcription factor or its downstream targets presents a novel therapeutic approach to treat ES. Research from our group demonstrated the anti-cancer activity of a small molecule, Tolfenamic acid (TA) in pediatric cancers, leukemia, neuroblastoma and medulloblastoma. TA has been shown to target transcription factor Sp1, which regulates the expression of several genes associated with cancer including survivin, a member of inhibitor of apoptosis protein (IAP) family and involved in chemoresistance. It is currently in phase I clinical trials for treating upper gastro-intestinal cancer patients along with radiation. In this study, we evaluated the therapeutic efficacy of TA alone and in combination with chemotherapeutic drugs, and its effect on the EWS-FLI1 downstream targets. ES cell lines CHLA9, CHLA10 and TC71 were treated with TA, Etoposide, Doxorubicin and Vincristine and cell viability was measured at 24 and 48h post-treatment. TA and chemotherapeutic drugs caused a dose and time dependent inhibition of cell proliferation. We then assessed the effects of TA in combination with these chemotherapeutic drugs. Results indicate that the combination treatment causes increased (2-3 fold) inhibition of cell viability compared to individual agents. To access the mode of action of combination therapy, cells were treated with TA and Vincristine and apoptosis was monitored at 48h post-treatment via western blot analysis (c-PARP), flow cytometry (Annexin V staining), and caspase 3/7 activity (Caspase3/7-Glo assay). We were able to demonstrate that the inhibition of cell viability in the combination treatment was the result of apoptotic mechanism as determined by increased cleavage of PARP and up-regulation of caspase 3/7activity. In addition we also studied the effects of TA on the regulation of EWS-FLI1 downstream targets NROB1, ID2, ROCO1 and LDB2 by real-time PCR. Results demonstrate that TA treatment leads to the downregulation of EWS-FLI1 genes. Taken together our results indicate that the combination of TA and chemotherapy drugs inhibits the proliferation of sarcoma cells. This investigation also suggests that the proposed combination therapy involving small molecule TA and standard chemotherapeutic agents could be a viable strategy for the treatment of Ewing sarcoma. Citation Format: Umesh T. Sankpal, Anish Ray, Leslie Tabor-Simecka, W Paul Bowman, Riyaz M. Basha. Effect of tolfenamic acid on the therapeutic efficacy of chemotherapeutic drugs used in the treatment of Ewing sarcoma. [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 2197. doi:10.1158/1538-7445.AM2015-2197