Vikalp Vishwakarma

Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis

Colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer-related mortality. Observed during CRC tumorigenesis is loss of post-transcriptional regulation of tumor-promoting genes such as COX-2, TNFα and VEGF. Overexpression of the RNA-binding protein HuR (ELAVL1) occurs during colon tumorigenesis and is abnormally present within the cytoplasm, where it post-transcriptionally regulates genes through its interaction with 3′UTR AU-rich elements (AREs). Here, we examine the therapeutic potential of targeting HuR using MS-444, a small molecule HuR inhibitor. Treatment of CRC cells with MS-444 resulted in growth inhibition and increased apoptotic gene expression, while similar treatment doses in non-transformed intestinal cells had no appreciable effects. Mechanistically, MS-444 disrupted HuR cytoplasmic trafficking and released ARE-mRNAs for localization to P-bodies, but did not affect total HuR expression levels. This resulted in MS-444-mediated inhibition of COX-2 and other ARE-mRNA expression levels. Importantly, MS-444 was well tolerated and inhibited xenograft CRC tumor growth through enhanced apoptosis and decreased angiogenesis upon intraperitoneal administration. In vivo treatment of MS-444 inhibited HuR cytoplasmic localization and decreased COX-2 expression in tumors. These findings provide evidence that therapeutic strategies to target HuR in CRC warrant further investigation in an effort to move this approach to the clinic.

Regulation of HuR structure and function by dihydrotanshinone-I

Abstract The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3′UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

Abstract 5540: Antitumor efficacy of synthetic formulations of active components in Arum palaestinum extracts in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) have a propensity for aggressive growth and metastasis, with high morbidity and low 5-year survival rates. Despite advances in treatment, the overall survival rate has only modestly improved over the past several years. Herbal tea extract from the root and leaves of Arum palaestinum has been used to prevent and facilitate treatment of cancer in Palestine. Very few studies have addressed the mechanisms and antitumor efficacy of this neutraceutical. Here we tested the antitumor efficacy of synthetic formulations of Arum palaestinum extract (GZ17-S, -05.00 and -06.02) in preclinical models of HNSCC. Our preliminary data demonstrates that these formulations have antitumor efficacy in several tumor types including HNSCC, lung and ovarian cancer cells. We hypothesized that GZ17-06.02 inhibits phosphorylation of key molecules that mitigate HNSCC progression and induce apoptotic cell death. Cytotoxicity of all the formulations was investigated on two HNSCC cell lines, OSC19 and UMSCC1. The GZ17-06.02 and -05.00 were found to be highly efficacious with low ED50 values (11.85 μg/ml and 16.40 μg/ml, respectively) for OSC19 and (10.86 μg/ml and 11.60 μg/ml, respectively) UMSCC1 cells at 72h. We determined the efficacy of GZ17 formulations in mitigating HNSCC invasion and migration. Among the formulations tested, GZ17-06.02 was most efficacious in significantly reducing invasion (37.33%) and migration (12.66%) of OSC19 cells compared to vehicle control (p Citation Format: Vikalp Vishwakarma, Kyle Crooker, Vusala Snyder, Jeffrey Straub, Caitlin Cole, Annie Lee, Lisa Stehno Bittel, Sufi Thomas. Antitumor efficacy of synthetic formulations of active components in Arum palaestinum extracts in head and neck squamous cell carcinoma. [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 5540. doi:10.1158/1538-7445.AM2015-5540

Potent Antitumor Effects of a Combination of Three Nutraceutical Compounds

Head and neck squamous cell carcinoma (HNSCC) is associated with low survival, and the current aggressive therapies result in high morbidity. Nutraceuticals are dietary compounds with few side effects. However, limited antitumor efficacy has restricted their application for cancer therapy. Here, we examine combining nutraceuticals, establishing a combination therapy that is more potent than any singular component, and delineate the mechanism of action. Three formulations were tested: GZ17-S (combined plant extracts from Arum palaestinum, Peganum harmala and Curcuma longa); GZ17-05.00 (16 synthetic components of GZ17-S); and GZ17-6.02 (3 synthetic components of GZ17S; curcumin, harmine and isovanillin). We tested the formulations on HNSCC proliferation, migration, invasion, angiogenesis, macrophage viability and infiltration into the tumor and tumor apoptosis. GZ17-6.02, the most effective formulation, significantly reduced in vitro assessments of HNSCC progression. When combined with cisplatin, GZ17-6.02 enhanced anti-proliferative effects. Molecular signaling cascades inhibited by GZ17-6.02 include EGFR, ERK1/2, and AKT, and molecular docking analyses demonstrate GZ17-6.02 components bind at distinct binding sites. GZ17-6.02 significantly inhibited growth of HNSCC cell line, patient-derived xenografts, and murine syngeneic tumors in vivo (P < 0.001). We demonstrate GZ17-6.02 as a highly effective plant extract combination and pave the way for future clinical application in HNSCC.

Characterization and protective efficacy of type three secretion proteins as a broadly protective subunit vaccine against Salmonella enterica serovars

ABSTRACT Nontyphoidal Salmonella enterica serotypes (NTS) are the leading cause of hospitalization and death due to foodborne illnesses. NTS are the costliest of the foodborne pathogens and cause ∼

Cancer-Associated Fibroblasts Drive Glycolysis in a Targetable Signaling Loop Implicated in Head and Neck Squamous Cell Carcinoma Progression

4 billion annually in health care costs. In Africa, new invasive NTS are the leading cause of bacteremia, especially in HIV-positive children and adults. Current vaccines against S. enterica are not broadly protective and most are directed at the typhoid-causing serotypes, not the NTS. All S. enterica strains require two type III secretion systems (T3SS) for virulence. The T3SS needle tip protein and the first translocator are localized to the T3SS needle tip and are required for pathogenesis of S. enterica. Collectively they are 95 to 98% conserved at the amino acid sequence level among all S. enterica strains. The Salmonella pathogenicity island 1 or 2 tip and first translocator proteins were genetically fused to produce the S1 and S2 fusion proteins, respectively, as potential vaccine candidates. S1 and S2 were then characterized using spectroscopic techniques to understand their structural and biophysical properties. Formulated at the proper pH, S1, S2, or S1 plus S2 (S1S2), admixed with adjuvant, was used to immunize mice followed by a lethal challenge with S. enterica serotype Typhimurium or S. enterica serotype Enteritidis. The S1S2 formulation provided the highest protective efficacy, thus demonstrating that an S1S2 subunit vaccine can provide broad, serotype-independent protection, possibly against all S. enterica serotypes. Such a finding would be transformative in improving human health.

Abstract B116: Mechanistic insights into the antitumor efficacy of nutraceutical GZ17-06.02, a highly effective formulation of Arum palaestinum extract, on head and neck squamous cell carcinoma

Despite aggressive therapies, head and neck squamous cell carcinoma (HNSCC) is associated with a less than 50% 5-year survival rate. Late-stage HNSCC frequently consists of up to 80% cancer-associated fibroblasts (CAF). We previously reported that CAF-secreted HGF facilitates HNSCC progression; however, very little is known about the role of CAFs in HNSCC metabolism. Here, we demonstrate that CAF-secreted HGF increases extracellular lactate levels in HNSCC via upregulation of glycolysis. CAF-secreted HGF induced basic FGF (bFGF) secretion from HNSCC. CAFs were more efficient than HNSCC in using lactate as a carbon source. HNSCC-secreted bFGF increased mitochondrial oxidative phosphorylation and HGF secretion from CAFs. Combined inhibition of c-Met and FGFR significantly inhibited CAF-induced HNSCC growth in vitro and in vivo (P < 0.001). Our cumulative findings underscore reciprocal signaling between CAF and HNSCC involving bFGF and HGF. This contributes to metabolic symbiosis and a targetable therapeutic axis involving c-Met and FGFR.Significance: HNSCC cancer cells and CAFs have a metabolic relationship where CAFs secrete HGF to induce a glycolytic switch in HNSCC cells and HNSCC cells secrete bFGF to promote lactate consumption by CAFs. Cancer Res; 78(14); 3769-82. ©2018 AACR.

733 - The RNA Binding Protein HUR Regulates Extracellular Vesicle Secretion in Colorectal Cancer

Although Arum palaestinum has been used as a natural prevention and treatment of cancer in Palestine for many years, few studies have examined its efficacy and mechanism of action. Chemically synthesized components of Arum palaestinum extract mixed in a ratio that was present in the natural extract were tested on head and neck squamous cell carcinoma (HNSCC), which affects annually 50,000 new people and causes 10,000 deaths a year in the US alone. We examined the effects of the nutraceutical on cell lines and patient-derived HNSCC tumors, and we delineated the mechanism of action. Among three formulations tested of Arum palaestinum (GZ17-S, a fortified version of the plant extract; GZ17-05.00, 16 components of the original plant extract; and GZ17-06.02, 3 components of the original plant extract), we found formulation GZ17-06.02 to be the most effective at reducing cancer cell proliferation, migration, and invasion. Further, GZ17-06.02 decreased angiogenesis in a tubule formation assay. When combined with a standard chemotherapeutic, cisplatin, GZ17-06.02 decreased cancer cell proliferation to a similar level of cisplatin, and enhanced the effect of cisplatin when used in combination. In comparison to radiotherapy, GZ17-06.02 performed better at reducing HNSCC cell proliferation than 3, 6, or 9 gy of radiation alone, and sequential treatment of HNSCC with radiation followed by GZ17-06.02 did not enhance the cytotoxic effects compared to GZ17-06.02 alone. We identified molecular signaling cascades inhibited by GZ17-06.02, including Src, ERK1/2, EGFR, AKT1/2/3, STAT-2, and Chk2, and we performed molecular docking analyses between GZ17-06.02 components and EGFR and AKT. We found that GZ17-06.02 components bind simultaneously at distinct binding sites of the kinase domains of EGFR and AKT. We found GZ17-06.02 induced significant apoptosis in HNSCC cells, increasing cleaved PARP, and activating caspase-3. Intratumoral injections of GZ17-06.02 were well tolerated in mice, and inhibited the growth and induced necrosis of HNSCC xenografts when injected intratumorally. In addition, orally administered GZ17-06.02 not only mitigated the growth of patient-derived HNSCC xenografts, but significantly decreased the tumor volume compared to the initially implanted tumor. Response to treatment correlated with a reduction in ERK1/2, which establishes it as an excellent biomarker for future clinical trials. Taken together, we demonstrate GZ17-06.02 as a highly effective nutraceutical, and pave the way for future clinical application in HNSCC. Citation Format: Jacob New, Vikalp Vishwakarma, Dhruv Kumar, Kyle Crooker, Vusala Snyder, Yelizaveta Shnayder, Kiran Kakarala, Terance T. Tsue, Douglas Girod, Lisa Stehno-Bittel, Sufi M. Thomas. Mechanistic insights into the antitumor efficacy of nutraceutical GZ17-06.02, a highly effective formulation of Arum palaestinum extract, on head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B116.