Xenograft Animal Model Research Articles

SPERT gene silencing inhibits the growth of human colon cancer xenograft tumor in nude mice via p38MAPK/HSP27 signaling pathway.

Colorectal cancer is one of the most common gastrointestinal malignancies and is also a disease of genetic heterogeneity. Our previous studies have shown that SPERT (sprermatid-associated protein) gene may be an underlying oncogene that is associated with the progression of the disease in colorectal cancer patients, and SPERT gene silencing can inhibit the proliferation of colorectal tumor cells and promote cell apoptosis. Here, we use the stably transfected human colorectal cancer cell line RKO to construct an animal xenograft model and study the effect of SPERT gene silencing on animal xenografts. The results showed that SPERT gene silencing can inhibit tumor growth in animals. In addition, through signaling pathway analysis, we found that the p38MAPK/HSP27 signaling pathway may be the molecular mechanism by which SPERT gene silencing inhibits the growth of xenograft tumors in nude mice. Combined with previous data, SPERT gene silencing has the same inhibitory effect on tumor growth in vitro and in vivo. These data suggest that SPERT gene may be a potential target for the treatment of colorectal cancer in clinic.

18F]Fluoro-DCP, a first generation PET radiotracer for monitoring protein sulfenylation in vivo

Redox metabolism plays essential functions in the pathology of cancer and many other diseases. While several radiotracers for imaging redox metabolism have been developed, there are no reports of radiotracers for in vivo imaging of protein oxidation. Here we take the first step towards this goal and describe the synthesis and kinetic properties of a new positron emission tomography (PET) [18F]Fluoro-DCP radiotracer for in vivo imaging of protein sulfenylation. Time course biodistribution and PET/CT studies using xenograft animal models of Head and Neck Squamous Cell Cancer (HNSCC) demonstrate its capability to distinguish between tumors with radiation sensitive and resistant phenotypes consistent with previous reports of decreased protein sulfenylation in clinical specimens of radiation resistant HNSCC. We envision further development of this technology to aid research efforts towards improving diagnosis of patients with radiation resistant tumors.

Liposome-encapsulated anthraquinone improves efficacy and safety in triple negative breast cancer

Breast cancer is the most common cancer among women and a leading cause of death worldwide. Triple negative breast cancer (TNBC) is a highly aggressive subtype which is the most challenging to treat. Due to heterogeneity and a lack of specific molecular targets, small molecule-based chemotherapy is the preferred course of treatment. However, these drugs have high toxicity due to off-target effects on healthy tissues, and tumors may develop resistance. Here, we present a polyethylene glycol-modified nanoscale liposomal formulation (LipoRV) of a new anthraquinone derivative which has potent effects on multiple TNBC cell lines. LipoRV readily inhibited the cell cycle, induced cell apoptosis, and reduced long-term proliferative potential of TNBC cells. In a xenograft animal model, LipoRV successfully cleared tumors and demonstrated a good safety profile, without detrimental effects on biochemical markers. Finally, RNA sequencing of LipoRV-treated TNBC cells was carried out, indicating that LipoRV may have immunomodulatory properties. These findings demonstrate that a liposomal anthraquinone-based molecule has excellent promise for TNBC therapy in the future.

Potential of antiviral drug oseltamivir for the treatment of liver cancer.

Liver cancer is a leading cause of cancer-related mortality globally. Since hepatitis virus infections have been strongly associated with the incidence of liver cancer, studies concerning the effects of antiviral drugs on liver cancer have attracted great attention in recent years. The present study investigated the effects of two anti-hepatitis virus drugs, lamivudine and ribavirin, and one anti-influenza virus drug, oseltamivir, on liver cancer cells to assess alternative methods for treating liver cancer. MTT assays, wound healing assays, Transwell assays, flow cytometry, immunoblotting, ELISA, immunofluorescence staining and a xenograft animal model were adopted to verify the effects of lamivudine, ribavirin and oseltamivir on liver cancer cells. Treatment with ribavirin and oseltamivir for 24 and 48 h significantly decreased the viability of both Huh-7 and HepG2 cells compared with that of THLE-3 cells in a dose-dependent manner. The subsequent investigations focused on oseltamivir, considering the more serious clinical adverse effects of ribavirin than those of oseltamivir. Significantly decreased migration and invasion were observed in both Huh-7 and HepG2 cells that were treated with oseltamivir for 24 and 48 h. In addition, oseltamivir significantly increased autophagy in Huh-7 cells, as revealed by the significantly higher ratios of LC3-II/LC3-I, increased expression of Beclin-1, and decreased expression of p62, whereas no significant increases in the expression of apoptosis-related proteins, including Apaf-1, cleaved caspase-3, and cleaved PARP-1, were detected. Notably, apoptosis and autophagy were significantly increased in HepG2 cells in the presence of oseltamivir, as revealed by the significant increases in the expression of Apaf-1, cleaved caspase-3, and cleaved PARP-1, the higher ratios of LC3-II/LC3-I, the increased expression of Beclin-1, and the decreased expression of p62. Additionally, significant inhibitory effects of oseltamivir on xenografted Huh-7 cells in athymic nude mice were observed. The present study, for the first time to the best of our knowledge, reported the differential effects of oseltamivir on inducing liver cancer cell death both in vitro and in vivo and may provide an alternative approach for treating liver cancer.

PERK-mediated IL8 secretion can enhance the migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells

BackgroundCancer stem cells (CSCs) drive tumor initiation and progression and participate in tumor chemoresistance. We recently discovered that oral squamous cell carcinoma (OSCC) cells that highly express CD10 (CD10H cells) present cancer stem cells (CSC)-associated characteristics, which, in turn, affect the tumor growth, epithelial-mesenchymal transition (EMT), and resistance to cisplatin. In this study, we further investigated this mechanism in vitro and in vivo. We hypothesized that IL8 might regulate migration, invasion, and cisplatin resistance of CD10-positive oral cancer cells through the ERK pathway.MethodsCD10 MicroBead Kit was used to select HN6 cells with high and low expression of CD10. The target protein IL8 was screened via protein chip assay. Lentiviral transduction and specific inhibitor were applied to investigate the signaling pathway. Real-time PCR, Western blot, and immunohistochemistry were used to analyze the mRNA and protein expression; transwell assay, spheroid formation assay, and cell viability assay were used to study the cell biological behavior in vitro; xenograft animal model was used to evaluate the tumor formation rate in vivo.ResultsOverexpression of CD10 promoted CSC-related genes expression and enhanced migration, invasion, spheroid formation, and chemoresistance in HN6 cells. Moreover, the overexpression of IL8 was detected in OSCC tumor tissue and cell lines (HN6 and CAL27) overexpressing CD10. IL8 secreted by CD10H HN6 promoted migration and invasion and restored tumor chemosensitivity via the p-ERK signaling pathway, while the inhibition of IL8 increased the chemosensitivity to cisplatin.ConclusionsIL8 secretion by CD10 positive cells promotes migration, invasion, and cisplatin resistance of OSCC via the p-ERK signaling pathway.

TRAP1 suppresses oral squamous cell carcinoma progression by reducing oxidative phosphorylation metabolism of Cancer-associated fibroblasts

BackgroundGlucose metabolism in cancer associated fibroblasts (CAFs) within the tumor microenvironment is a material and energy source for tumorigenesis and tumor development. However, the characteristics and important regulatory mechanisms of glucose metabolism in fibroblasts associated with oral squamous cell carcinoma (OSCC) are still unknown.MethodsWe successfully isolated, cultured, purified and identified CAFs and normal fibroblasts (NFs). Cell culture, immunohistochemistry (IHC) and CCK8, flow cytometry, Seahorse XF Analyzer, MitoTracker assay, western blotting (WB), transmission electron microscope, Quantitative real-time PCR (qPCR), immunofluorescence (IF), and Label-free quantitative proteomics assay, animal xenograft model studies and statistical analysis were applied in this study.ResultsWe demonstrated that the proliferation activity of CAFs was significantly enhanced as compared to NFs, while the apoptosis rate was significantly decreased. CAFs in OSCC preferentially use oxidative phosphorylation (OXPHOS) rather than glycolysis. Moreover, CAFs showed stronger maximal respiration, a larger substantial mitochondrial spare respiratory capacity (SRC) and higher adenosine triphosphate (ATP) production capacity than NFs. The results of mitotracker green fluorescence staining showed that compared with NFs, CAFs exhibited stronger green fluorescence. The results of WB showed the expression level of Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) obviously increased in CAFs compared to NFs. These results confirmed that CAFs have greater mitochondrial activity and function than NFs. Furthermore, Label-free quantitative proteomics assays showed that both ATP synthase subunit O (ATP5O) and tumor necrosis factor receptor-associated protein 1 (TRAP1) are important differentially expressed proteins in the mitochondria of CAFs/NFs. Overexpression of TRAP1 in CAFs increased basal oxygen consumption rate (OCR), maximal respiration, ATP production and SRC. In vivo, overexpression TRAP1 expression in CAFs suppress tumor growth.ConclusionTaken together, the results indicated that TRAP1 is an important regulatory molecule of CAFs glucose metabolism and promotes OSCC progression by regulating the OXPHOS of CAFs.

FAK Regulates VEGFR2 Expression and Promotes Angiogenesis in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) remains a significant clinical challenge because of its high vascularity and metastatic and recurrent rates. Tumor angiogenesis is considered an important mediator in the regulation of tumor cell survival and metastasis in TNBC. Angiogenesis is induced by the binding of vascular endothelial growth factor to vascular endothelial growth factor receptor 2 (VEGFR2). Focal adhesion kinase (FAK) plays an important role in regulating various cell functions in normal and cancer cells. Previous studies have focused on investigating the function of endothelial FAK in tumor cell angiogenesis. However, the association between tumor FAK and VEGFR2 in tumor angiogenesis and the possible mechanisms of this remain unclear. In this study, we used a public database and human specimens to examine the association between FAK and VEGFR2. At the same time, we verified the association between FAK and VEGFR2 through several experimental methods, such as quantitative real-time polymerase chain reaction, Western blotting, and next-generation sequencing. In addition, we used the endothelial cell model, zebrafish, and xenograft animal models to investigate the role of FAK in TNBC angiogenesis. We found that FAK and VEGFR2 were positively correlated in patients with TNBC. VEGFR2 and several other angiogenesis-related genes were regulated by FAK. In addition, FAK regulated VEGFR2 and VEGF protein expression in TNBC cells. Functional assays showed that FAK knockdown inhibited endothelial tube formation and zebrafish angiogenesis. An animal model showed that FAK inhibitors could suppress tumor growth and tumor vascular formation. FAK promotes angiogenesis in TNBC cells by regulating VEGFR2 expression. Therefore, targeting FAK could be another antiangiogenic strategy for TNBC treatment.

AKF-D52, a Synthetic Phenoxypyrimidine-Urea Derivative, Triggers Extrinsic/Intrinsic Apoptosis and Cytoprotective Autophagy in Human Non-Small Cell Lung Cancer Cells.

Simple SummaryWe previously reported the antiproliferative effects of a phenoxypyridine urea derivative. In this study, we aimed to investigate the antiproliferative effects of 1-(3,5-dimethoxyphenyl)-3-(4-(3-methoxyphenoxy)-2-((4-morpholinophenyl)amino)pyrimidin-5-yl)urea (AKF-D52) in non-small cell lung cancer cells. We found that (i) AKF-D52 induces apoptosis in caspase-dependent and caspase-independent pathways; (ii) AKF-D52-induced apoptosis is caused by the clustering of a death-inducing signaling complex and mitochondrial-dependent signaling; (iii) AKF-D52 induces cytoprotective autophagy, and pre-treatment with an autophagy inhibitor enhances the apoptotic effect of AKF-D52; and (iv) AKF-D52-induced apoptosis and autophagy are attenuated by the reactive oxygen species (ROS) scavenger α-tocopherol. Furthermore, AKF-D52 suppressed tumor growth in a xenograft mouse model. Collectively, our findings regarding the efficacy and molecular mechanisms of AKF-D52 identify this compound as a potential therapeutic agent for the treatment of lung cancer.Previously, we discovered that 1-(3,5-dimethoxyphenyl)-3-(4-(3-methoxyphenoxy)-2-((4-morpholinophenyl)amino)pyrimidin-5-yl)urea (AKF-D52), a synthetic phenoxypyrimidine urea derivative, acts as a growth inhibitor of various cancer cell types. In this study, we elucidated the antiproliferative properties of AFK-D52 and underlying mechanisms in non-small cell lung cancer (NSCLC) cells and an A549 xenograft animal model. AKF-D52 was found to induce both caspase-dependent and -independent apoptotic cell death. Furthermore, the mitochondrial component of the AKF-D52-induced apoptosis mechanism involves a reduction in mitochondrial membrane potential and regulation in B cell lymphoma-2 family protein expression. Moreover, AKF-D52 activates the extrinsic pathway through up-regulated expression of death receptor 3 and Fas and then the formation of a death-inducing signaling complex. AKF-D52 also induced autophagy by increasing acidic vesicular organelle formation and microtubule-associated protein 1A/1B-light chain 3-II levels and reducing p62 levels. Notably, pretreatment with autophagy inhibitors enhanced AKF-D52-induced cell death, indicating that the induced autophagy is cytoprotective. AKF-D52 treatment also triggered reactive oxygen species (ROS) production in NSCLC cells, whereas the antioxidant α-tocopherol abolished AKF-D52-induced cell death. In a xenograft lung cancer mouse model, AKF-D52 administration attenuated tumor growth by inducing apoptosis and autophagy in tumor tissues. Collectively, our data indicate that AKF-D52-induced ROS production plays a role in mediating apoptosis and cytoprotective autophagy in NSCLC.

Troponin C-1 Activated by E2F1 Accelerates Gastric Cancer Progression via Regulating TGF-β/Smad Signaling.

Troponin C-1 (TNNC1) has been previously characterized as an oncogenic gene. This study aimed to reveal the roles of TNNC1 in gastric cancer and the potential underlying mechanisms. TNNC1 siRNAs and TNNC1 overexpression plasmid were used to alter its expression in AGS, MKN45, and HGC-27 cells. CCK-8 assay, colony formation, EdU assay, flow cytometry, transwell assay, and scratch test were conducted to measure the phenotype changes. In vivo effects of TNNC1 silence were confirmed by using a xenograft mouse model. Bioinformatics analysis was conducted to screen out the transcription factor and downstream signaling of TNNC1. TNNC1 was highly expressed in gastric cancer tissues and cell lines, and its expression was associated with poor prognosis. TNNC1 silence suppressed the proliferation, migration, and invasion of AGS and MKN45 cells. However, TNNC1 silence induced apoptosis by mediating the cleavage of caspase-3 and caspase-9. Overexpression of TNNC1 in HGC-27 cells led to the contrary effects. The anti-tumor effects of TNNC1 silence were also confirmed in a xenograft animal model. E2F1 was validated as an upstream transcription factor of TNNC1. Effects of TNNC1 silence on AGS cell migration and invasion were attenuated by E2F1 overexpression. Besides, TGF-β/Smad was a downstream signaling pathway of TNNC1. The anti-tumor impacts of TNNC1 silence were weaken by SB431542 (a specific inhibitor of TGF-β signaling) while accelerated by TGF-β. TNNC1 activated by E2F1 functioned as an oncogenic gene through regulating TGF-β/Smad signaling. TNNC1 was suggested as a potential molecular drug target of gastric cancer.

A Phase 1, Open-Label, Dose-Escalation Study of the Safety and Efficacy of Anti-CD38 Antibody Drug Conjugate (STI-6129) in Patients with Relapsed or Refractory Multiple Myeloma

Background: With the advent of several new agents in the treatment of multiple myeloma, including proteasome inhibitors (PIs), immunomodulatory drugs (IMiDs), and anti-CD38 monoclonal antibodies (mAb), the overall survival (OS) has significantly improved in the past two decades. However, most patients become refractory to currently available therapies along the disease trajectory. The median OS in triple-class-refractory patients (i.e., anti-CD 38 mAb, PIs, and IMiDs) is 6 months (Gandhi et al. Leukemia. 2019). Although BCMA-targeted therapies are a major advance in such patients, none of them are curative thus far, with the median progression-free survival (PFS) ranging from 3 months in BCMA-antibody drug conjugates (ADCs) to approximately 1 year in BCMA chimeric antigen receptor T-cell (CAR T-cell) therapies. Furthermore, unique toxicities of BCMA-targeted agents such as ocular toxicity (ADCs), cytokine release syndrome, or neurotoxicity (CAR Ts and BiTEs) may preclude their use in many patients. Hence, there is a critical unmet need in patients with triple-class-refractory myeloma.The STI-6129 ADC is produced by conjugating STI-5171, a fully human anti-CD38 mAb, with a covalently bound tubulin inhibitor, duostatin 5.2 (DUO-5.2), using a proprietary linker technology with a 3:1 drug-antibody ratio. STI-6129 binds to different CD38 epitopes than daratumumab. Upon binding to CD38, STI-6129 ADC is internalized by the cancer cell and undergoes lysosomal degradation, releasing DUO-5.2. This in turn leads to G2-phase cell cycle arrest, followed by caspase 3/7-dependent apoptosis and cell death. In studies on cynomolgus monkeys, the serum level of DUO-5.2 remained undetectable at all doses except at the highest dose of 4.5 mg/kg, indicating low likelihood of off-target toxicity. In vitro studies of primary human plasma cells, human tumor models, and animal xenograft models have demonstrated target elimination of CD38-positive human plasma cells by STI-6129. Importantly, STI-6129 has demonstrated cytotoxic activity in human multiple myeloma cells isolated from daratumumab-refractory patients (Figure 1). Hence, further investigation on clinical activity of STI-6129 is warranted.Study Design: The 38-ADC-RRMM-101 study is a two-stage, multicenter, open-label, dose-finding phase 1/2 trial. The phase 1 trial is designed to identify the recommended phase 2 dose (RP2D) of STI-6129 by assessing safety, preliminary efficacy, and pharmacokinetics in patients with relapsed/refractory (RR) myeloma. The phase 2a stage of the trial will be a single-arm study to investigate the efficacy of STI-6129 in an expansion cohort of RR myeloma patients. Up to 25 patients will be enrolled in the phase 1 stage and 30 in the phase 2 stage of the study. The key inclusion criteria are the following: (a) RR myeloma with at least 3 prior lines of therapy in addition to being refractory to at least 1 PI, 1 IMiD, and 1 anti-CD 38 mAb; (b) measurable disease. The key exclusion criteria are: (a) receipt of the last dose of any anti-CD mAb within 90 days; (b) grade ≥3 neuropathy or grade 2 neuropathy with pain; (c) current history of CTCAE grade 3 muscle paresis, eyelid conditions, glaucoma, or any other ocular disorder that is CTCAE grade 2; (d) estimated creatinine clearance <60 ml/min; (e) left ventricular ejection fraction<40%. The primary endpoint of phase 1 stage of the study is safety, particularly any dose limiting toxicities. The primary endpoint of the phase 2 part of the study is overall response rate as per IMWG criteria. The dosing cohorts in the dose escalation phase will range from 0.25 mg/kg to 3.68 mg/kg. Given the potential for on-target off-tumor toxicity due to expression of CD38 on non-clonal plasma cells and other hematopoietic cells, blood counts and immunoglobulin levels will be closely monitored. While neurotoxicity or ocular toxicity were not observed with STI-6129 in animal models, including non-human primates, such event will be considered as adverse events of special interest (AESI) and comprehensive neurology and ocular (including slit lamp) examinations will be performed at baseline and study completion, and at any AESI. STI-6129 will be administered intravenously once in a 4-week cycle, with the intention being to treat patients until disease progression or unacceptable toxicity. [Display omitted] DisclosuresYan: Sorrento Therapeutics: Current Employment. Royal: Sorrento Therapeutics: Current Employment.

APR-246 Reveals a Therapeutic Potential Via Triggering Different Cell Death Mechanisms in Diffuse Large B Cell Lymphoma

Background: Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy and is characterized by its pronounced genetic and clinical heterogeneity. Although the first-line therapy (R-CHOP) improves the curability of patients with DLBCL, nearly 40% remainder ultimately undergo relapsed or refractory disease. Emerging evidence has shown that TP53 mutations correlate with the recurrence and progression of DLBCL. APR-246, also known as PRIMA-1MET, can reactivate the trans-activation of TP53 mutants by facilitating their DNA binding to target genes, making it a promising therapeutic compound for mutated TP53 carcinomas. Although APR-246 in combination with other chemicals has been applied in clinical trials for non-Hodgkin lymphoma (NHL), the efficacy and the underlying molecular mechanisms of APR-246 on DLBCL remain unclear. The arms are to investigate the TP53 mutations and the correlation of mutated TP53 with the prognosis. Furthermore, we evaluated the effect of APR-246 on DLBCL in vitro and in vivo.Methods: Multiple datasets, including the Gene Expression Omnibus (GEO) and cBioPortal, were searched for available data for DLBCL. For evaluation of APR-246 effect on DLBCL in vitro, ten DLBCL cell lines harbouring different and representative molecular properties, especially distinct TP53 mutation status, were tested. To validate the therapeutic effect of APR-246 in vivo, NSG mice were injected subcutaneously with the DLBCL cells with TP53 mutations to establish a xenograft animal model, and then these mice received the APR-246 administration. The cell viability of DLBCL was measured post addition of APR-246. Pharmaceutical inhibition of different cell death pathways was applied to elucidate the mechanisms by which APR-246 functions.Results: Total 2204 patients with DLBCL were evaluated, of which nearly 15% contained TP53 mutations. The missense mutation of TP53 was up to 76% and mutations occurring in the DNA binding domain (DBD) was about 90%. Patients with TP53 mutations had poor OS (p=0.0118). Further, we found that patients with TP53 mutations in GCB and UNC subtypes exhibited inferior OS (p=0.043; p=0.049, respectively), but no in ABC subtype. Notably, TP53 single mutations located in the DBD (exon 5-8) led to an unfavorable survival (p=0.0263), while patients carrying TP53 multiple mutations and single mutations in other domains exhibited no difference in survival time than those with wild-type TP53. APR-246 induced the cell death in a dose dependent manner for all DLBCL cell lines. DLBCL cells with TP53 missense mutations in the DBD were the most sensitive to APR-246 than those with intact or other types of mutated TP53. APR-246 mediated the cell death via p53-dependent ferritinophagy for DLBCL cells with TP53 missense mutations in the DBD. In addition, APR-246 also induced the ferroptosis for other DLBCL cells harbouring wild type TP53 and other forms of TP53 mutations.Conclusions: Nearly 15% of patients with DLBCL harbouring TP53 mutations had inferior prognosis than those with wild-type TP53. Patients with TP53 single mutations occurred in the DBD (exon 5-8) showed poorer prognosis than those with TP53 multiple mutations and single mutations in other domains. APR-246 induced the cell death of DLBCL through different mechanisms, depend on different forms of TP53 mutations, which provided an evidence for clinical application of APR-246 in the treatment of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tipifarnib enhances anti-EGFR activity of cetuximab in non-HRas mutated head and neck squamous cell carcinoma cancer (HNSCC)

ObjectiveTo test the potential ability of tipifarnib to impair proliferation and to enhance the activity of the EGFR inhibitor cetuximab in wild-type H-Ras HNSCC, which accounts for the majority of HNSCC. Materials and MethodsCell growth, apoptosis and signaling changes in HNSCC cells following tipifarnib exposure in vitro were assessed by SRB, colony formation assay, annexin V staining and Western blot, respectively. A patient-derived xenograft (PDX) animal model was adopted to evaluate the efficacy of tipifarnib in vivo with and without cetuximab. ResultsTreatment of wild-type H-Ras HNSCC cell lines in vitro with tipifarnib reduced cell growth and increased levels of defarnesylated H-Ras in a dose-dependent manner. In a PDX mouse model, treatment with single-agent tipifarnib led to only near-significant growth inhibition. The addition of cetuximab resulted in increased anti-proliferative effect both in culture and in PDX models, which was also mirrored by Western blot and apoptosis assay results. ConclusionTipifarnib has only a moderate ability to slow tumor growth as a single agent in HNSCC with wild type H-Ras, despite specifically inhibiting the farnesyltransferase upon which the function of H-Ras depends. The combination of cetuximab and tipifarnib appears to enhance the anti-proliferative effect of single-agent tipifarnib and marginally enhance that of single agent cetuximab. These findings deserve further evaluation.

FAM83A promotes proliferation and metastasis via Wnt/\u03b2-catenin signaling in head neck squamous cell carcinoma

This research aimed to investigate the expression and function of FAM83A in the proliferation and metastasis in head and neck squamous cell carcinoma (HNSCC). FAM83A mRNA and protein expressions in HNSCC were detected in primary HNSCC samples and cell lines. The associations between FAM83A expression and clinicopathologic variables were evaluated through tissue microarrays. Besides, FAM83A knockdown and overexpression cell lines were constructed to assess cell growth and metastasis in vitro and the relationship between FAM83A and epithelial-mesenchymal transition (EMT). Furthermore, two models of xenograft tumors in nude mice were used to assess the tumorigenicity and metastasis ability of FAM83A in vivo. In the present study, overexpression of FAM83A in HNSCC samples was significantly associated with tumor size, lymph node status and clinical tumor stages. Mechanically, FAM83A could promote HNSCC cell growth and metastasis by inducing EMT via activating Wnt/β-catenin signaling pathway. Rescue experiment demonstrated the inhibition of β-catenin could counteract the function of FAM83A. Also, the FAM83A knockdown could suppress tumor growth and distant metastasis in the xenograft animal models of HNSCC. In conclusion, this study identifies FAM83A as an oncogene of HNSCC. This study provides new insights into the molecular pathways that contribute to EMT in HNSCC. We revealed a previously unknown FAM83A-Wnt–β-catenin signaling axis involved in the EMT of HNSCC. There may be a potential bi-directional signaling loop between FAM83A and Wnt/β-catenin signaling pathway in HNSCC.

Considerations for Whole-Slide Analysis of Murine Xenografts Experiments.

Histochemistry of tumor sections is a widely employed technique utilized to examine cell death in preclinical xenograft animal models of cancer. However, this is under the assumption that tumors are homogeneous, leading to practices such as automatic cell counting across the entire section. We have noted that in our experiments the core of the tumor is largely or partially necrotic, and lacks evidence of vascularization (in contrast to the outer areas of the tumor). We note that this can bias and confound immunohistochemical analyses that do not take care to sample areas of interest in a way to take this into account. Design-based stereology with image analysis techniques is an alternative process that could be used to measure the volume of the necrotic region compared to the volume of the whole tumor.

Albumin Binder-Conjugated Fibroblast Activation Protein Inhibitor Radiopharmaceuticals for Cancer Therapy.

Fibroblast activation protein (FAP) has become an attractive target for diagnosis and therapy, and a series of FAP inhibitor (FAPI)-based radiotracers has been developed and had excellent performance for diagnosis outcomes in clinical applications. Yet, their fast clearance and insufficient tumor retention have hampered their further clinical application in cancer treatment. In this study, we developed 2 albumin binder-conjugated FAPI radiotracers, TEFAPI-06 and TEFAPI-07. They were derived from FAPI-04 and were optimized by conjugating 2 types of well-studied albumin binders, 4-(p-iodophenyl) butyric acid moiety (TEFAPI-06) and truncated Evans blue moiety (TEFAPI-07), to try to overcome the above limitations at the expense of prolonging the blood circulation. Methods: TEFAPI-06 and TEFAPI-07 were synthesized and labeled with 68Ga, 86Y, and 177Lu successfully. A series of cell assays was performed to identify the binding affinity and FAP specificity invitro. PET imaging, SPECT imaging, and biodistribution studies were performed to evaluate the pharmacokinetics in pancreatic cancer patient-derived xenograft (PDX) animal models. The cancer treatment efficacy of 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 were evaluated in pancreatic cancer PDX-bearing mice. Results: The binding affinities (dissociation constants) to FAP of 68Ga-TEFAPI-06 and 68Ga-TEFAPI-07 were 10.16 ± 2.56 nM and 7.81 ± 2.28 nM, respectively, which were comparable with that of 68Ga-FAPI-04. Comparative PET imaging of HT-1080-FAP and HT-1080 tumor-bearing mice and a blocking study showed the FAP-targeting ability invivo of these 2 tracers. Compared with 177Lu-FAPI-04, PET imaging, SPECT imaging, and biodistribution studies of TEFAPI-06 and TEFAPI-07 demonstrated their remarkably enhanced tumor accumulation and retention, respectively. Notable tumor growth inhibition by 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 were observed, whereas the control group and the group treated by 177Lu-FAPI-04 showed a slight therapeutic effect. Conclusion: Two albumin binder-conjugated FAPI radiopharmaceuticals have been developed and evaluated invitro and invivo. Significantly improved tumor uptake and retention were observed, compared with the original FAPI tracer. Both 177Lu-TEFAPI-06 and 177Lu-TEFAPI-07 showed remarkable growth inhibition of PDX tumors, whereas the side effects were almost negligible, demonstrating that these radiopharmaceuticals are promising for further clinical translational studies.

Selenite-induced ROS/AMPK/FoxO3a/GABARAPL-1 signaling pathway modulates autophagy that antagonize apoptosis in colorectal cancer cells

Previous studies have shown that selenium possessed chemotherapeutic effect against multiple malignant cancers, inducing diverse stress responses including apoptosis and autophagy. Selenite was previously shown to induce apoptosis and autophagy in colorectal cancer cells. However, the relationship between selenite-induced apoptosis and autophagy was not fully understood. Our results revealed a pro-survival role of selenite-induced autophagy against apoptosis in colorectal cancer cells. Real-time PCR array of autophagy-related genes showed that GABARAPL-1 was significantly upregulated in colorectal cancer cells, which was confirmed by western blot and immunofluorescence results. Knockdown of GABARAPL-1 significantly inhibited selenite-induced autophagy and enhanced apoptosis. Furthermore, we found that selenite-induced upregulation of GABARAPL-1 was caused by upregulated p-AMPK and FoxO3a level. Their interaction was correlated with involved in regulation of GABARAPL-1. We observed that activation and inhibition of AMPK influenced both autophagy and apoptosis level via FoxO3a/ GABARAPL-1 signaling, implying the pro-survival role of autophagy against apoptosis. Importantly, we corroborated these findings in a colorectal cancer xenograft animal model with immunohistochemistry and western blot results. Collectively, these results show that sodium selenite could induce ROS/AMPK/FoxO3a/GABARAPL-1-mediated autophagy and downregulate apoptosis in both colorectal cancer cells and colon xenograft model. These findings help to explore sodium selenite as a potential anti-cancer drug in clinical practices.

Double-edged Swords: Diaryl pyrazoline thiazolidinediones synchronously targeting cancer epigenetics and angiogenesis

In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds 13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments. It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.) also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot.

EBV latent membrane proteins promote hybrid epithelial-mesenchymal and extreme mesenchymal states of nasopharyngeal carcinoma cells for tumorigenicity.

EBV-encoded LMPs are consistently detected in nasopharyngeal carcinoma (NPC). Recent evidence suggests potential roles of LMP1 and LMP2A in Epithelial-to-mesenchymal transition (EMT) process in NPC. EMT engages in the generation and maintenance of cancer stem cells (CSCs) and confers on cancer cells increased tumor-initiating and metastatic potential, and higher resistance to anticancer therapies. However, how LMP1 and LMP2A regulate the EMT process to generate cells with different EMT states and its implications for tumor progression remain unclear. Here we report that LMP1 and LMP2A promote EMT that drives NPC cells from the epithelial-like state (E) (CD104+, CD44low) to epithelial-mesenchymal hybrid (E/M) state (CD104+, CD44high). Furthermore, LMP2A possesses an additional function in stabilizing LMP1 and increasing the level of LMP1 in NPC cells. The elevated LMP1 further forces the EMT to generate extreme-mesenchymal (xM) state cells (CD104-, CD44high). To define the tumorigenic features of cancer stem cells at different states in the EMT spectrum, E, E/M and xM subpopulations were isolated and tested for tumorigenic capability in a tumor xenograft animal model. We found that the cells with E/M phenotypes possess the highest tumor initiating capacity. However, the xM subpopulation exhibits increased vasculogenic mimicry, a hallmark of metastatic cancers. Taken together, coordinated action of LMP1 and LMP2A generates an array of intermediate subpopulations in the EMT spectrum that are responsible for distinct tumorigenic features of NPC such as tumor-initiation, vasculogenesis, and metastasis.

Abstract 1039: Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1

Abstract Background: Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths in the US. In this malignancy, 5-fluorouracil (5FU)-based treatment remains the mainstay as first-line chemotherapy. Unfortunately, ~50-60% of patients treated with 5FU eventually develop resistance, leading to increased mortality. Hence, a better understanding of the molecular mechanisms underlying 5FU-resistance will lead to development of more effective therapeutic strategies. Our previous studies have shown that, andrographis, a botanical with diverse biological activities, synergistically enhanced 5FU-induced pro-apoptotic effects by mediating Wnt/β-catenin pathways. However, a more comprehensive knowledge of andrographis-mediated synergism with 5-FU remains largely unclear. Methods: We first established 5FU resistant isogenic cell lines of HCT116 and SW480 CRC cells (HCT116 5FUR and SW480 5FUR). To determine the synergistic anti-tumor effects of andrographis and 5FU, we performed cell viability, proliferation and colony formation assays by treating parental and 5FUR cells with andrographis, 5FU and their combination. To explore the molecular mechanisms responsible for the synergistic anti-tumor efficacy, we next performed gene expression microarrays to identify candidate pathways and genes that mediated anti-tumor efficacy of andrographis in 5FUR cells. We analyzed an expression profiling dataset from CRC patients (GSE52735) who either responded or did not respond to 5FU. We also studied the andrographis-mediated synergism with 5FU in a xenograft animal model, and in 3D patient-derived tumor organoids. Results: Combined treatment with andrographis and 5FU exhibited significantly superior effects on cell viability (P&amp;lt;0.01), proliferation (P&amp;lt;0.001) and colony formation capacity (P&amp;lt;0.001), compared to treatment with individual treatments. In line with our in vitro findings, the combined treatment also significantly reduced tumor growth in mice xenografts (P&amp;lt;0.0001) as well as patient-derived tumoroids (P&amp;lt;0.001). By analyzing gene expression profiling data from cell lines and CRC patients, we identified that overexpression of DKK1 gene was a critical event for 5FU resistance in CRC. Moreover, qRT-PCR and western blotting data revealed that andrographis resulted in downregulation of 5FU-induced DKK1 overexpression accompanied with the synergistic anti-tumor effects in animal xenografts (P&amp;lt;0.001) and patient-derived tumor organoids (P&amp;lt;0.001), which were reversed following siRNA-mediated silencing of DKK1 (P&amp;lt;0.01). Conclusions: Our data provide novel evidence for andrographis-mediated anti-tumor synergism with 5-FU through DKK1, highlighting that in addition to its preventive efficacy, andrographis may serve as a potential adjunctive treatment to conventional chemotherapeutic drugs in patients with CRC. Citation Format: Yinghui Zhao, Chuanxin Wang, Ajay Goel. Andrographis enhances 5-fluorouracil-induced anti-tumor effect in colorectal cancer through inhibition of DKK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1039.

Abstract 2389: LINC00901 regulates glucose metabolism in pancreatic cancer

Abstract Dysregulation of metabolism is one of the cancer hallmarks. However, little is known whether long coding RNAs (lncRNAs) play a role in the regulation of metabolism in pancreatic ductal adenocarcinoma (PDAC). In the present study, we focused on lncRNA LINC00901 because a high level of LINC00901 is associated with the poor outcome of PDAC patients. Functional studies showed that LINC00901 promotes PDAC cell proliferation and invasion, whereas knockout (KO) of LINC00901 impairs these abilities of PDAC cells. Furthermore, the xenograft animal model suggested that LINC00901 promotes tumorigenesis. To determine the underlying mechanism of LINC00901-mediated tumorigenesis, we performed RNA-seq analysis in LINC00901 overexpression or KO cells. Gene Set Enrichment Analysis (GSEA) analysis showed that gene sets involved in glucose biological processes such as ‘‘MYC targets”, “Glycolysis” and “Gluconeogenesis” are significantly enriched in the LINC00901 overexpression group, whereas these genes are down-regulated in the LINC00901 KO group. The top-scoring genes recurring in the three gene sets include key glycolysis-related genes, lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and enolase 1 (ENO1), all of which were validated by qRT-PCR and western blot. Moreover, we found that LINC00901 upregulates c-Myc by stabilizing c-Myc RNA. Together, these results suggest that LINC00901 is an oncogenic lncRNA capable of regulating glucose metabolism, leading to PDAC development. Experiments are underway to determine how LINC00901 regulates c-Myc RNA stability in pancreatic cancer cells. Citation Format: Wan-Xin Peng, Liu Yang, Yin-Yuan Mo. LINC00901 regulates glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2389.