Xenografts In Athymic Nude Mice Research Articles

Pre-clinical Efficacy and Safety Pharmacology of PEGylated Recombinant Human Endostatin.

This study aimed to outline the pre-clinical efficacy and safety pharmacology of PEGylated recombinant human endostatin (M2ES) according to the requirements of new drug application. The purity of M2ES was evaluated by using silver staining. Transwell migration assay was applied to detect the bioactivity of M2ES in vitro. The antitumor efficacy of M2ES was evaluated in an athymic nude mouse xenograft model of pancreatic cancer (Panc-1) and gastric cancer (MNK45). BALB/C mice were treated with different doses of M2ES (6, 12 and 24 mg/kg) intravenously, both autonomic activity and cooperative sleep were monitored before and after drug administration. The apparent molecular weight of M2ES was about 50 kDa, and the purity was greater than 98%. Compared with the control group, M2ES significantly inhibits human micro-vascular endothelial cells (HMECs) migration in vitro. Notably, weekly administration of M2ES showed a significant antitumor efficacy when compared with the control group. Treatment of M2ES (24mg/kg or below) showed no obvious effect on both autonomic activity and hypnosis. On the basis of the pre-clinical efficacy and safety pharmacology data of M2ES, M2ES can be authorized to carry out further clinical studies.

A novel NR4A2-HuR axis promotes pancreatic cancer growth and tumorigenesis that is inhibited by NR4A2 antagonists.

Pancreatic ductal adenocarcinoma (PDAC) patients' express higher levels of the orphan Nuclear Receptor 4A2 (NR4A2, NURR1) compared to normal pancreas and NR4A2 is a prognostic factor for patient survival. Knockdown of NR4A2 by RNA interference (RNAi) inhibited cell proliferation, invasion, and migration. RNA sequencing performed in NR4A2(+/+) and NR4A2(-/-) MiaPaCa2 cells demonstrated that NR4A2 played a significant role in cellular metabolism. Human antigen R (HuR) and isocitrate dehydrogenase 1 (IDH1) were identified as NR4A2 target genes. HuR is a pro-oncogenic RNA binding protein and silencing of HuR by RNAi significantly downregulated expression of NR4A2. Expression of HuR and IDH1 were significantly downregulated after treatment with NR4A2 inverse agonist, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane resulting in significant inhibition of tumor growth in an athymic nude mouse xenograft model. This study demonstrates that NR4A2 and HuR regulate genes and signaling pathways that enhance tumorigenesis and targeting NR4A2 and HuR expression with an NR4A2 inverse agonist represents a novel regimen for treating PDAC.

Radiosensitization by Hyperthermia Critically Depends on the Time Interval

Background and PurposeHyperthermia is a potent sensitizer of radiotherapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiotherapy are still under debate. Experimental designWe investigated the interval and sequence in vitro in cervical cancer cell lines and patient-derived organoids, in vivo in SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. ResultsAll three approaches confirmed that shortening the interval between hyperthermia and radiotherapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis, 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥ 80 min) to 54% (interval <80 min). In vitro and in vivo results showed that the sequence of hyperthermia and radiotherapy did not affect the outcome. ConclusionsShortening the interval between hyperthermia and radiotherapy significantly improves treatment outcomes. The sequence of hyperthermia and radiotherapy (before or after) does not seem to matter.

Inhibition of Monoacylglycerol Lipase Decreases Angiogenic Features of Endothelial Cells via Release of Tissue Inhibitor of Metalloproteinase-1 from Lung Cancer Cells.

Despite the well-described anticarcinogenic effects of endocannabinoids, the influence of the endocannabinoid system on tumor angiogenesis is still debated. In the present study, conditioned medium (CM) from A549 and H358 lung cancer cells treated with ascending concentrations of the monoacylglycerol lipase (MAGL) inhibitor JZL184 and 2-arachidonoylglycerol (2-AG), a prominent MAGL substrate, caused a concentration-dependent reduction in human umbilical vein endothelial cell (HUVEC) migration and tube formation compared with CM from vehicle-treated cancer cells. Comparative experiments with MAGL inhibitors JW651 and MJN110 showed the same results. On the other hand, the angiogenic properties of HUVECs were not significantly altered by direct stimulation with JZL184 or 2-AG or by exposure to CM of JZL184- or 2-AG-treated non-cancerous bronchial epithelial cells (BEAS-2B). Inhibition of HUVEC migration and tube formation by CM of JZL184- and 2-AG-treated A549 cells was abolished in the presence of the CB1 antagonist AM-251. Increased release of tissue inhibitor of metalloproteinase-1 (TIMP-1) from JZL184- or 2-AG-stimulated A549 or H358 cells was shown to exert an antiangiogenic effect on HUVECs, as confirmed by siRNA experiments. In addition, JZL184 caused a dose-dependent regression of A549 tumor xenografts in athymic nude mice, which was associated with a decreased number of CD31-positive cells and upregulation of TIMP-1-positive cells in xenograft tissue. In conclusion, our data suggest that elevation of 2-AG by MAGL inhibition leads to increased release of TIMP-1 from lung cancer cells, which mediates an antiangiogenic effect on endothelial cells.

Bromocriptine monotherapy overcomes prostate cancer chemoresistance in preclinical models

Chemoresistance is a major obstacle in the clinical management of metastatic, castration-resistant prostate cancer (PCa). It is imperative to develop novel strategies to overcome chemoresistance and improve clinical outcomes in patients who have failed chemotherapy. Using a two-tier phenotypic screening platform, we identified bromocriptine mesylate as a potent and selective inhibitor of chemoresistant PCa cells. Bromocriptine effectively induced cell cycle arrest and activated apoptosis in chemoresistant PCa cells but not in chemoresponsive PCa cells. RNA-seq analyses revealed that bromocriptine affected a subset of genes implicated in the regulation of the cell cycle, DNA repair, and cell death. Interestingly, approximately one-third (50/157) of the differentially expressed genes affected by bromocriptine overlapped with known p53-p21- retinoblastoma protein (RB) target genes. At the protein level, bromocriptine increased the expression of dopamine D2 receptor (DRD2) and affected several classical and non-classical dopamine receptor signal pathways in chemoresistant PCa cells, including adenosine monophosphate-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor kappa B (NF-κB), enhancer of zeste homolog 2 (EZH2), and survivin. As a monotherapy, bromocriptine treatment at 15 mg/kg, three times per week, via the intraperitoneal route significantly inhibited the skeletal growth of chemoresistant C4-2B-TaxR xenografts in athymic nude mice. In summary, these results provided the first preclinical evidence that bromocriptine is a selective and effective inhibitor of chemoresistant PCa. Due to its favorable clinical safety profiles, bromocriptine could be rapidly tested in PCa patients and repurposed as a novel subtype-specific treatment to overcome chemoresistance.

A powerful drug combination strategy targeting BRAF-mutant melanoma.

3152 Background: BRAF inhibitors, such as vemurafenib and dabrafenib, are effective in treating patients with melanoma harboring (V600E) BRAF mutations. These drugs block the activity of the mutated BRAF protein, which impacts the development and progression of melanoma. Despite initial efficacy, drug resistance reliably occurs leading to disease relapse. Here we show how BRAF inhibitors cause metabolic reprogramming in melanoma cells to achieve drug resistance and promote cell survival. Previously we demonstrated that wild-type isocitrate dehydrogenase 1 (wtIDH1) supports mitochondrial function and maintains redox homeostasis in melanoma, and that an FDA-approved mutant IDH1 inhibitor, ivosidenib (AG-120), is actually a potent wtIDH1 inhibitor. Thus, we hypothesized that wtIDH1 inhibition would synergize with BRAF inhibition. Methods: Metabolomic profiling using LC-MS determined the impact of BRAF inhibition on cellular metabolism. Cell viability was assessed by PicoGreen in drug combination assays. Synergism was calculated using the Bliss-Independence dose-response model for drug interaction. In vivo modeling of AG-120 in combination with BRAF inhibition used melanoma cell xenografts in athymic nude mice for tumor volume analyses, and survival studies were performed in both immunocompetent C57BL/6J mice (murine YUMM1.7 cells) and immunocompromised athymic nude mice (human A375 cells). Results: Metabolomics data suggest that BRAF-mutated melanoma cells treated with BRAF inhibitors increased oxidative metabolism and mitochondrial dependence (i.e., survival adaptations). These alterations suggest an opportunity for mitochondrial inhibitors to improve drug efficacy. Targeting wtIDH1 with AG-120 in combination with dabrafenib significantly reduced melanoma cell viability. A positive synergy score and Bliss score greater than 1 indicate that the combined treatment was more effective in reducing cell viability than either treatment alone. Further, the combination of AG-120 and dabrafenib was the most effective in vivo, reducing tumor growth in the BRAF-mutated melanoma xenograft model, and improving mouse survival compared to monotherapy controls. Mice tolerated combination therapy with no reduction in body weight observed. Conclusions: Our findings indicate that dual inhibition of mutated BRAF and wild-type IDH1 represents a novel treatment strategy for BRAF-mutated melanomas, with potential for application to other BRAF-mutated cancers (e.g., colon, hepatobiliary, thyroid, etc.).

Abstract 3701: Targeting wild-type IDH1 enhances chemosensitivity in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality in the United States. The poor prognosis is in part due to development of chemotherapy resistance, despite improvements in multiagent regimens. Our previous work demonstrated that oxidative stress plays an important role in drug resistance. Wild-type isocitrate dehydrogenase 1 (IDH1) is an important enzyme that generates cytosolic NADPH to maintain redox homeostasis and protect cancer cells from oxidative damage. Additionally, we demonstrated that ivosidenib (AG-120), an FDA-approved mutant IDH1 inhibitor, is actually a potent inhibitor of wild-type IDH1, under low magnesium and nutrient levels that are present in the tumor microenvironment. Methods: We evaluated IDH1 expression in PDAC using The Cancer Genome Atlas (TCGA). Cell viability was assessed by Trypan blue and PicoGreen in drug combination assays. Cellular reactive oxygen species (ROS) levels were determined by the DCFDA method. To further assess the therapeutic potential of AG-120 in combination with chemotherapy, tumor volume analyses were performed using patient-derived xenografts (PDX) in athymic nude mice, and survival studies were performed in C57BL/6J mice transplanted with orthotopic murine pancreatic cancer. Results: Analysis of TCGA data indicated that IDH1 is overexpressed in pancreatic cancer tumors. Treatment of MiaPaca2 and Panc1 cancer cells with 5- fluorouracil (5-FU) induced expression of wild-type IDH1 in vitro. Short-term cell viability data demonstrated that targeting IDH1 with AG-120 when combined with DNA-damaging agents (5-FU, oxaliplatin) had a synergistic effect with a positive synergy score and Bliss score greater than 1. Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for both Panc1 and MiaPaCa-2 cells when 5-FU was combined with AG-120, as compared to single-agent controls. Inhibiting IDH1 impairs the ability of pancreatic cancer cells to scavenge ROS levels, enhances chemotherapy-induced apoptosis in pancreatic cancer cells via ROS-mediated damage in vitro. Both PDX tumor volume studies and overall survival analyses revealed that the combination of these AG-120 and chemotherapy synergistically enhanced anti-tumor activity and doubled the survival benefits as compared to single-agent alone. Conclusion: IDH1 plays a critical role in tumorigenesis and chemoresistance in pancreatic cancer. Our data demonstrate that IDH1 inhibition with AG-120 may enhance chemotherapy efficacy and represents an important area for future investigation in the form of clinical trials. Citation Format: Mehrdad Zarei, Omid Hajihassani, Jonathan J. Hue, Hallie J. Graor, Arian Hajihassani, Alexander W. Loftus, Luke D. Rothermel, Jordan M. Winter. Targeting wild-type IDH1 enhances chemosensitivity in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3701.

Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways.

Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

The role of Cannabidiol and tetrahydrocannabivarin to overcome doxorubicin resistance in MDA-MB-231 xenografts in athymic nude mice

The significant resistance to currently available chemotherapeutics makes treatment for TNBC a key clinical concern. Herein, we studied the anti-cancer potentials of synthetic cannabidiol (CBD) and Tetrahydrocannabivarin (THCV) when used alone or in combination with doxorubicin (DOX) against MDA-MB-231 resistant cells. Pre-treatment with CBD and THCV significantly increased the cytotoxicity of DOX in MDA-MB-231 2D and 3D cultures that were DOX-resistant. Transcriptomics and Proteomics studies revealed that CBD and THCV, by downregulating PD-L1, TGF-β, sp1, NLRP3, P38-MAPK, and upregulating AMPK induced apoptosis leading to improved DOX's chemosensitivity against DOX resistant MDA-MB-231 tumors in BALB/c nude mice. CBD/THCV in combination with DOX significantly inhibited H3k4 methylation and H2K5 acetylation as demonstrated by western blotting and RT-PCR. Based on these findings, CBD and THCV appear to counteract histone modifications and their subsequent effects on DOX, resulting in chemo-sensitization against MDA-MB-231 resistant cancers.

Combined Transcriptomic and Proteomic Profiling to Unravel Osimertinib, CARP-1 Functional Mimetic (CFM 4.17) Formulation and Telmisartan Combo Treatment in NSCLC Tumor Xenografts.

The epidermal growth factor receptor (EGFR) is highly expressed in many non-small cell lung cancers (NSCLC), necessitating the use of EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatments. Osimertinib (OSM), a third-generation TKI, is routinely used in clinics, but T790M mutations in exon 20 of the EGFR receptor lead to resistance against OSM, necessitating the development of more effective therapeutics. Telmisartan (TLM), OSM, and cell cycle and apoptosis regulatory protein 1 (CARP-1) functional mimetic treatments (CFM4.17) were evaluated in this study against experimental H1975 tumor xenografts to ascertain their anti-cancer effects. Briefly, tumor growth was studied in H1975 xenografts in athymic nude mice, gene and protein expressions were analyzed using next-generation RNA sequencing, proteomics, RT-PCR, and Western blotting. TLM pre-treatment significantly reduced the tumor burden when combined with CFM-4.17 nanoformulation and OSM combination (TLM_CFM-F_OSM) than their respective single treatments or combination of OSM and TLM with CFM 4.17. Data from RNA sequencing and proteomics revealed that TLM_CFM-F_OSM decreased the expression of Lamin B2, STAT3, SOD, NFKB, MMP-1, TGF beta, Sox-2, and PD-L1 proteins while increasing the expression of AMPK proteins, which was also confirmed by RT-PCR, proteomics, and Western blotting. According to our findings, the TLM_CFM-F_OSM combination has a superior anti-cancer effect in the treatment of NSCLC by affecting multiple resistant markers that regulate mitochondrial homeostasis, inflammation, oxidative stress, and apoptosis.

Plant Alkaloid Tetrandrine Is a Nuclear Receptor 4A1 Antagonist and Inhibits Panc-1 Cell Growth In Vitro and In Vivo.

The orphan nuclear receptor 4A1 (NR4A1) is highly expressed in human pancreatic cancer cells and exerts pro-oncogenic activity. In a previous study, we demonstrated that fangchinoline (FCN), a natural inhibitor of nuclear NR4A1, induces NR4A1-dependent apoptosis in human pancreatic cancer cells. In this study, we evaluated FCN and its structural analogs (berbamine, isotetrandrine, tetrandrine, and tubocurarine) for their inhibitory effects on NR4A1 transactivity, and confirmed that tetrandrine (TTD) showed the highest inhibitory effect in pancreatic cancer cells. Moreover, in a tryptophan fluorescence quenching assay, TTD directly bound to the ligand binding domain (LBD) of NR4A1 with a KD value of 10.60 μM. Treatment with TTD decreased proliferation and induced apoptosis in Panc-1 human pancreatic cancer cells in part through the reduced expression of the Sp1-dependent anti-apoptotic gene survivin and induction of ROS-mediated endoplasmic reticulum stress, which are the well-known NR4A1-dependent proapoptotic pathways. Furthermore, at a dose of 25 mg/kg/day, TTD reduced tumor growth in an athymic nude mouse xenograft model bearing Panc-1 cells. These data show that TTD is an NR4A1 antagonist and that modulation of the NR4A1-mediated pro-survival pathways is involved in the antitumor effects of TTD.

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.

BAP1 Downregulates NRF2 Target Genes and Exerts Anti-Tumorigenic Effects by Deubiquitinating KEAP1 in Lung Adenocarcinoma.

KELCH-ECH-associated protein 1 (KEAP1) is an adaptor protein of Cullin 3 (CUL3) E3 ubiquitin ligase that targets a redox sensitive transcription factor, NF-E2-related factor 2 (NRF2). BRCA1-associated protein 1 (BAP1) is a tumor suppressor and deubiquitinase whose mutations increase the risk of several types of familial cancers. In the present study, we have identified that BAP1 deubiquitinates KEAP1 by binding to the BTB domain. Lentiviral transduction of BAP1 decreased the expression of NRF2 target genes, suppressed the migration and invasion, and sensitized cisplatin-induced apoptosis in human lung adenocarcinoma (LUAD) A549 cells. Examination of the lung tissues in KrasG12D/+ mice demonstrated that the level of Bap1 and Keap1 mRNAs progressively decreases during lung tumor progression, and it is correlated with NRF2 activation and the inhibition of oxidative stress. Supporting this observation, lentiviral transduction of BAP1 decreased the growth of A549 xenografts in athymic nude mice. Transcriptome analysis of human lung tissues showed that the levels of Bap1 mRNA are significantly higher in normal samples than LUAD samples. Moreover, the expression of Bap1 mRNA is associated with a better survival of LUAD patients. Together, our study demonstrates that KEAP1 deubiquitination by BAP1 is novel tumor suppressive mechanism of LUAD.

Role of extracellular vesicles secretion in paclitaxel resistance of prostate cancer cells.

Aim: The development of chemotherapy resistance is the major obstacle in the treatment of advanced prostate cancer (PCa). Extracellular vesicles (EVs) secretion plays a significant role among different mechanisms contributing to chemoresistance. Hence, inhibition of EVs release may increase the efficacy of chemotherapeutic drugs against PCa. Methods: Paclitaxel (PTX) resistant PCa cells (PC3-R and DU145-R) were treated with GW4869, a known exosome biogenesis inhibitor. EVs were isolated from the conditioned media by ExoQuick-based precipitation method and characterized for concentration and size distribution by nanoparticle tracking analysis. The effect of GW4869 treatment on the survival and growth of PCa cells was assessed by MTT, and colony formation assays in vitro, and ectopic PC3-R xenografts in male athymic nude mice in vivo. The effect of other EV biogenesis inhibitors, imipramine and dimethyl amiloride (DMA), treatment was also analyzed on the survival of PC3-R cells. Results: GW4869 (10-20 µM) treatment of PTX resistant PCa cells significantly reduced the release of small EVs (50-100 nm size range) while increasing the release of larger EVs (> 150 nm in size), and inhibited their clonogenicity. Moreover, GW4869 (5-20 µM) treatment (24-72h) significantly inhibited the survival of PC3-R cells in a dose-dependent manner. We observed a similar growth inhibition with both imipramine (5-20 µg/mL) and DMA (5-20 µg/mL) treatment in PC3-R cells. Furthermore, GW4869 treatment (IP) in mice bearing PC3-R xenografts significantly reduced the tumor weight (65% reduction, P = 0.017) compared to the vehicle-treated control mice without causing any noticeable toxicity. Conclusion: Inhibiting the release of EVs could sensitize the resistant PCa cells to chemotherapy.

Flavonoids kaempferol and quercetin are nuclear receptor 4A1 (NR4A1, Nur77) ligands and inhibit rhabdomyosarcoma cell and tumor growth

BackgroundFlavonoids exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with KD values of 3.1 and 0.93 μM, respectively.MethodsThe activities of kaempferol and quercetin were determined in direct binding to NR4A1 protein and in NR4A1-dependent transactivation assays in Rh30 and Rh41 rhabdomyosarcoma (RMS) cells. Flavonoid-dependent effects as inhibitors of cell growth, survival and invasion were determined in XTT and Boyden chamber assays respectively and changes in protein levels were determined by western blots. Tumor growth inhibition studies were carried out in athymic nude mice bearing Rh30 cells as xenografts.ResultsKaempferol and quercetin bind NR4A1 protein and inhibit NR4A1-dependent transactivation in RMS cells. NR4A1 also regulates RMS cell growth, survival, mTOR signaling and invasion. The pro-oncogenic PAX3-FOXO1 and G9a genes are also regulated by NR4A1 and, these pathways and genes are all inhibited by kaempferol and quercetin. Moreover, at a dose of 50 mg/kg/d kaempferol and quercetin inhibited tumor growth in an athymic nude mouse xenograft model bearing Rh30 cells.ConclusionThese results demonstrate the clinical potential for repurposing kaempferol and quercetin for clinical applications as precision medicine for treating RMS patients that express NR4A1 in order to increase the efficacy and decrease dosages of currently used cytotoxic drugs.

Melatonin inhibits proliferation, migration, and invasion by inducing ROS-mediated apoptosis via suppression of the PI3K/Akt/mTOR signaling pathway in gallbladder cancer cells.

Background: Melatonin is an indolic compound mainly secreted by the pineal gland and plays a vital role in the regulation of circadian rhythms and cancer therapy. However, the effects of melatonin in gallbladder cancer (GBC) and the related mechanism remain unknown.Methods: In this study, the antitumor activity of melatonin on gallbladder cancer was explored both in vitro and in vivo. After treatment with different concentrations of melatonin, the cell viability, migration, and invasion of gallbladder cancer cells (NOZ and GBC-SD cells) were evaluated by CCK-8 assay, wound healing, and Transwell assay.Results: The results showed that melatonin inhibited growth, migration, and invasion of gallbladder cancer cells. Subsequently, the assays suggested that melatonin significantly induced apoptosis in gallbladder cancer cells and altered the expression of the apoptotic proteins, including Bax, Bcl-2, cytochrome C, cleaved caspase-3, and PARP. Besides, the intracellular reactive oxygen species (ROS) was found to be upregulated after melatonin treatment in gallbladder cancer cells. Melatonin was found to suppress the PI3K/Akt/mTOR signaling pathway in a time-dependent manner by inhibiting the phosphorylation of PI3K, Akt, and mTOR. Treatment with N-acetyl-L-cysteine (NAC) or 740 Y-P remarkably attenuated the antitumor effects of melatonin in NOZ and GBC-SD cells. Finally, melatonin suppressed the growth of GBC-SD cells in an athymic nude mice xenograft model in vivo.Conclusions: Our study revealed that melatonin could induce apoptosis by suppressing the PI3K/Akt/mTOR signaling pathway. Therefore, melatonin might serve as a potential therapeutic drug in the future treatment of gallbladder cancer.

In vivo tumor growth inhibition by Solanum tuberosum aspartic protease 3 (StAP3) treatment

Solanum tuberosum aspartic Proteases (StAPs) show selective plasma membrane permeabilization, inducing cytotoxicity of cancer cells versus normal cells in vitro. Herein, we aimed to evaluate both StAP3 systemic toxicity and antitumoral activity against human melanoma in vivo. The toxicity of a single high dose of StAP3 (10 µg/g body weight, intraperitoneally) was assessed in a Balb/c mice model. Subcutaneous A375 human melanoma xenografts in athymic nude (nu/nu) mice were induced. Once tumors developed (mean larger dimension = 3.8 ± 0.09 mm), mice were StAP3-treated (6 µg/g body weight, subcutaneously under the tumor at a single dose). For both models, controls were treated with physiologic saline solution. StAP3-treated mice showed a significant inhibition of tumor growth (p < 0.05) compared with controls. No signs of toxicity were detected in StAP3-treated mice in both models. These results suggest the potential of these plant proteases as anticancer agents.

Characterization of genetically defined sporadic and hereditary type 1 papillary renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) is not a single disease but is made up of several different histologically defined subtypes that are associated with distinct genetic alterations which require subtype specific management and treatment. Papillary renal cell carcinoma (pRCC) is the second most common subtype after conventional/clear cell RCC (ccRCC), representing ~20% of cases, and is subcategorized into type 1 and type 2 pRCC. It is important for preclinical studies to have cell lines that accurately represent each specific RCC subtype. This study characterizes seven cell lines derived from both primary and metastatic sites of type 1 pRCC, including the first cell line derived from a hereditary papillary renal carcinoma (HPRC)‐associated tumor. Complete or partial gain of chromosome 7 was observed in all cell lines and other common gains of chromosomes 16, 17, or 20 were seen in several cell lines. Activating mutations of MET were present in three cell lines that all demonstrated increased MET phosphorylation in response to HGF and abrogation of MET phosphorylation in response to MET inhibitors. CDKN2A loss due to mutation or gene deletion, associated with poor outcomes in type 1 pRCC patients, was observed in all cell line models. Six cell lines formed tumor xenografts in athymic nude mice and thus provide in vivo models of type 1 pRCC. These type 1 pRCC cell lines provide a comprehensive representation of the genetic alterations associated with pRCC that will give insight into the biology of this disease and be ideal preclinical models for therapeutic studies.

The Axin2-snail axis promotes bone invasion by activating cancer-associated fibroblasts in oral squamous cell carcinoma

BackgroundIn bone-invasive oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAFs) infiltrate into bony tissue ahead of OSCC cells. In the present study, we aimed to investigate the role of the Axin2-Snail axis in the biological behaviour of CAFs and bone invasion in OSCC.MethodsThe clinicopathological significance of Axin2 and Snail expression was investigated by immunohistochemistry in an OSCC cohort containing 217 tissue samples from patients with long-term follow-up. The influence of the Axin2-Snail axis on the biological behaviour of OSCC cells and CAFs was further investigated both in vitro and in vivo.ResultsAxin2 expression was significantly associated with Snail expression, the desmoplasia status, and bone invasion in patients with OSCC. In multivariate analysis, lymph node metastasis, desmoplasia, Axin2 expression, and Snail expression were independent poor prognostic factors in our cohort. Consistent with these findings, OSCC cells demonstrated attenuated oncogenic activity as well as decreased expression of Snail and various cytokines after Axin2 knockdown in vitro. Among the related cytokines, C-C motif chemokine ligand 5 (CCL5) and interleukin 8 (IL8) demonstrated a strong influence on the biological behaviour of CAFs in vitro. Moreover, both the desmoplastic reaction and osteolytic lesions in the calvaria were predominantly decreased after Axin2 knockdown in OSCC cells in vivo using a BALB/c athymic nude mouse xenograft model.ConclusionsOncogenic activities of the Axin2-Snail axis are not limited to the cancer cells themselves but rather extend to CAFs via regulation of the cytokine-mediated cancer-stromal interaction, with further implications for bone invasion as well as a poor prognosis in OSCC.

Abstract B64: Targeting the pH regulators in the tumor microenvironment for ovarian cancer treatment

Abstract The high mortality rate in ovarian cancer (OVCA) is currently the most pressing concern despite the research advancements. It is still not clear how to effectively treat OVCA patients to prevent a relapse or to treat the relapse tumors that display a drug-resistant profile. To address this issue, this study targets a uniquely expressed pH regulator in the tumor microenvironment as a strategy to treat ovarian cancer. In order to survive the metabolic acidosis, cancer cells overexpress pH regulators on their surface to pump out excess protons/lactic acid. This, in turn, acidifies the tumor microenvironment and promotes tumor invasion/chemoresistance. The a2-Vacuolar-ATPases (V-ATPases) are the major pH-sensing unit of the V-ATPase proton pumps and are abundantly expressed on the surface of ovarian cancer cells. To establish the clinical importance, our findings show that a2-V-ATPases are overexpressed in all morphologic subtypes and stages of human OVCA and are absent on normal ovarian tissues. We further examined the therapeutic potential of monoclonal antibody against a2-V-ATPase (V0a2-mab) in controlling tumor growth in the preclinical mouse model. In vivo V0a2-mab treatment (300μg in PBS; i.p) resulted in a significantly delayed tumor growth compared to control, with no apparent in vivo toxicity in athymic nude mice xenograft model. The delayed tumor growth was attributed to high immune infiltration with increased leukocyte population and an enhanced iNOS expression compared to control mice. These results show that V-ATPases are overexpressed in the tumor microenvironment and their inhibition impairs OVCA growth by interfering with tumor acidification that enhances the antitumor responses. In conclusion, the study demonstrates that targeting V-ATPase pH regulators is an effective treatment strategy in ovarian cancer. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sylvia Schneiderman, Valerie E. Riehl, Alexandria N. Young, Shayna Levine, Safaa Ibrahim, Mahmood Bilal, Alice Gilman-Sachs, Kenneth D. Beaman. Targeting the pH regulators in the tumor microenvironment for ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B64.