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Background Circular RNAs (circRNAs) act as gene expression regulators and are involved in cancer progression. However, their functions have not been sufficiently investigated in nasopharyngeal carcinoma (NPC). Methods The expression profiles of circRNAs in NPC cells within different metastatic potential were reanalyzed. Quantitative reverse transcription PCR and in situ hybridization were used to detect the expression level of circPVT1 in NPC cells and tissue samples. The association of expression level of circPVT1 with clinical properties of NPC patients was evaluated. Then, the effects of circPVT1 expression on NPC metastasis were investigated by in vitro and in vivo functional experiments. RNA immunoprecipitation, pull-down assay and western blotting were performed to confirm the interaction between circPVT1 and β-TrCP in NPC cells. Co-immunoprecipitation and western blotting were performed to confirm the interaction between β-TrCP and c-Myc in NPC cells. Results We find that circPVT1, a circular RNA, is significantly upregulated in NPC cells and tissue specimens. In vitro and in vivo experiments showed that circPVT1 promotes the invasion and metastasis of NPC cells. Mechanistically, circPVT1 inhibits proteasomal degradation of c-Myc by binding to β-TrCP, an E3 ubiquiting ligase. Stablization of c-Myc by circPVT1 alters the cytoskeleton remodeling and cell adhesion in NPC, which ultimately promotes the invasion and metastasis of NPC cells. Furthermore, c-Myc transcriptionally upregulates the expression of SRSF1, an RNA splicing factor, and recruits SRSF1 to enhance the biosynthesis of circPVT1 through coupling transcription with splicing, which forms a positive feedback for circPVT1 production. Conclusions Our results revealed the important role of circPVT1 in the progression of NPC through the β-TrCP/c-Myc/SRSF1 positive feedback loop, and circPVT1 may serve as a prognostic biomarker or therapeutic target in patients with NPC.

Cancer chemoprevention is the most effective approach to control cancer in the population. Despite significant progress, chemoprevention has not been widely adopted because agents that are safe tend to be less effective and those that are highly effective tend to be toxic. Thus, there is an urgent need to develop novel and effective chemopreventive agents, such as mitochondria-targeted agents, that can prevent cancer and prolong survival. Mitochondria, the central site for cellular energy production, have important functions in cell survival and death. Several studies have revealed a significant role for mitochondrial metabolism in promoting cancer development and progression, making mitochondria a promising new target for cancer prevention. Conjugating delocalized lipophilic cations, such as triphenylphosphonium cation (TPP+), to compounds of interest is an effective approach for mitochondrial targeting. The hyperpolarized tumor cell membrane and mitochondrial membrane potential allow for selective accumulation of TPP+ conjugates in tumor cell mitochondria versus those in normal cells. This could enhance direct killing of precancerous, dysplastic, and tumor cells while minimizing potential toxicities to normal cells.

International audience; Combined treatment targeting mitochondria may improve the efficacy of lung cancer chemoprevention. Here, mitochondria-targeted honokiol (Mito-HNK), an inhibitor of mitochondrial complex I and STAT3 phosphorylation, and mitochondria-targeted lonidamine (Mito-LND), an inhibitor of mitochondrial complexes I/II and AKT/mTOR/p70S6K signaling, were evaluated for their combinational chemopreventive efficacy on mouse lung carcinogenesis. All chemopreventive treatments began one-week post-carcinogen treatment and continued daily for 24 weeks. No evidence of toxicity (including liver toxicity) was detected by monitoring serum levels of alanine aminotransferase and aspartate aminotransferase enzymes. Mito-HNK or Mito-LND treatment alone reduced tumor load by 56% and 48%, respectively, whereas the combination of Mito-HNK and Mito-LND reduced tumor load by 83%. To understand the potential mechanism(s) of action for the observed combinatorial effects, single-cell RNA sequencing was performed using mouse tumors treated with Mito-HNK, Mito-LND, and their combination. In lung tumor cells, Mito-HNK treatment blocked the expression of genes involved in mitochondrial complex ǀ, oxidative phosphorylation, glycolysis, and STAT3 signaling. Mito-LND inhibited the expression of genes for mitochondrial complexes I/II, oxidative phosphorylation, and AKT/mTOR/p70S6K signaling in lung tumor cells. In addition to these changes, a combination of Mito-HNK with Mito-LND decreased arginine and proline metabolism, N-glycan biosynthesis, and tryptophan metabolism in lung tumor cells. Our results demonstrate that Mito-LND enhanced the antitumor efficacy of Mito-HNK, where both compounds inhibited common targets (oxidative phosphorylation) as well as unique targets for each agent (STAT3 and mTOR signaling). Therefore, the combination of Mito-HNK with Mito-LND may present an effective strategy for lung cancer chemoprevention.

Circular RNAs (circRNAs) play an essential role in tumorigenesis and development. However, they have rarely been investigated in nasopharyngeal carcinoma (NPC). This study aimed to investigate the role of circRNA in the invasion and metastasis of NPC. We screened and verified the high expression of circSETD3 in NPC cell lines using RNA sequencing (RNA-Seq) and verified the results of NPC biopsy samples using real-time quantitative polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH). In vivo and in vitro experiments indicated that circSETD3 could promote NPC cell invasion and migration. We compared the proteomic data of NPC cells before and after the overexpression or knockdown of circSETD3 in combination with bioinformatics prediction and experimental verification. It was found that circSETD3 competitively adsorbs to miR-615-5p and miR-1538 and negates their inhibitory effect on MAPRE1 mRNA, thereby upregulating the expression of MAPRE1. The upregulated MAPRE1 then inhibits the acetylation of α-tubulin, promotes the dynamic assembly of microtubules, and enhances the invasion and migration capabilities of NPC cells. The results of this study suggest that circSETD3 is a novel molecular marker and a potential target for NPC diagnosis and treatment.

Background Magnolia extract (ME) is known to inhibit cancer growth and metastasis in several cell types in vitro and in animal models. However, there is no detailed study on the preventive efficacy of ME for oral cancer, and the key components in ME and their exact mechanisms of action are not clear. The overall goal of this study is to characterize ME preclinically as a potent oral cancer chemopreventive agent and to determine the key components and their molecular mechanism(s) that underlie its chemopreventive efficacy. Methods The antitumor efficacy of ME in oral cancer was investigated in a 4-nitroquinoline-1-oxide (4NQO)-induced mouse model and in two oral cancer orthotopic models. The effects of ME on mitochondrial electron transport chain activity and ROS production in mouse oral tumors was also investigated. Results ME did not cause detectable side effects indicating that it is a promising and safe chemopreventive agent for oral cancer. Three major key active compounds in ME (honokiol, magnolol and 4-O-methylhonokiol) contribute to its chemopreventive effects. ME inhibits mitochondrial respiration at complex I of the electron transport chain, oxidizes peroxiredoxins, activates AMPK, and inhibits STAT3 phosphorylation, resulting in inhibition of the growth and proliferation of oral cancer cells. Conclusion Our data using highly relevant preclinical oral cancer models, which share histopathological features seen in human oral carcinogenesis, suggest a novel signaling and regulatory role for mitochondria-generated superoxide and hydrogen peroxide in suppressing oral cancer cell proliferation, progression, and metastasis.

Due to their various biological activities that are beneficial to human health and antitumor effect, flavonoid compounds have attracted much attention in recent years. Hydrous icaritin (HICT) was such a flavonoid that can inhibit the growth of breast cancer and cancer stem cells. In order to overcome the insolubility problem, HICT was fabricated into nanorods (NRs) through anti-solvent precipitation in this paper using D-α tocopherol acid polyethylene glycol succinate and sodium oleate as a co-stabilizer meanwhile using the mixture of ethanol and acetone (1:2, v/v) as the organic solvent. The obtained HICT NRs showed an average particle size 222.0 nm with a small polydispersity index value of 0.124 and a high zeta potential of – 49.5 mV. HICT NRs could maintain similar particle size in various physiological medium and could be directly lyophilized without the addition of any cytoprotectants and then reconstituted into a colloidal system of similar size. The resultant HICT NRs had a high drug loading content of 55.6% and released HICT in a steady and constant pattern. MTT assay indicated NRs enhanced HICT’s antitumor activity to ninefold against MCF-7 breast carcinoma cells. In vivo studies demonstrated oral administration free HICT had almost no tumor inhibitory effect while HICT NRs showed a tumor inhibition rate of 47.8%. When intravenously injected, HICT NRs displayed similar therapeutic efficacy to paclitaxel injections (70.4% vs. 74.5%, TIR). This may be partly due to the high accumulation of the injected HICT NRs in tumor ranking only second to that in the liver but much higher than in other organs. These results demonstrated that HICT NRs could be a promising antitumor agent for the treatment of breast cancer in clinic.

Top2A is a key enzyme involved in DNA replication and is a therapeutic target for several cancer types including breast cancer. Overexpression of Top2A has been observed in both human and mouse triple-negative breast cancer (TNBC). The present study evaluated both immunogenicity and antitumor efficacy of a newly formulated multi-peptide vaccine targeting multiple epitopes of the Top2A protein. Top2A-specific MHC II epitopes with optimal binding affinity were identified using a combined scoring system, which predicted their potential to elicit a Th1 immune response. The formulated vaccine contained top three Top2A peptides, which elicited the strongest immunologic response and showed 100% sequence homology between human and mouse. Anti-tumor efficacy of the Top2A vaccine was initially evaluated in a syngeneic TNBC mouse model, in which pre-graft preventive vaccination was associated with significantly decreased tumor growth as compared to the adjuvant controls. The Top2A peptide vaccine exhibited striking efficacy in a genetically engineered TNBC mouse model (C3(1)/Tag), reducing tumor burden by >90% when compared with adjuvant alone. Splenocytes collected from vaccinated animals showed a robust immunologic response to the immunizing peptides. There were no overt toxicities observed with the Top2A vaccination. To explore potential mechanisms underlying the anti-tumor response induced by Top2A vaccine treatment, scTCR-seq of tumors in both control and Top2A vaccine groups revealed new T cell clones as a consequence of Top2A vaccination. Furthermore, in vitro stimulation of these splenocytes by the vaccinated Top2A peptides resulted in the secretion of cytokines indicative of Th1 responses but with minimal secretion of Th2-related cytokines. Our data indicate that the newly developed multi-peptide Top2A vaccine is immunogenic and efficacious in the prevention of TNBC development and progression in vivo. Citation Format: Sang Beom Lee, Jing Pan, Donghai Xiong, Katie Palen, Bryon Johnson, Jeffrey E. Green, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. Immunoprevention of triple negative breast cancer by TOP2A derived peptide vaccination [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA015.

Background Circular RNAs play an important role in tumor genesis and progression, but they have not been sufficiently studied in patients with nasopharyngeal carcinoma (NPC). Methods The circular RNA, circCAMSAP1, was screened in NPC cells by RNA sequencing analysis. The expression of circCAMSAP1 in NPC tissues was examined by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization. Wound-healing, transwell, MTT and flow cytometry assays, and nude mouse tumor models were used to explore the effect of circCAMSAP1 on proliferation and metastasis of NPC in vitro or in vivo. The downstream proteins regulated by circCAMSAP1 were screened using mass spectrometry. The interaction between circCAMSAP1 and the SERPINH1 mRNA was identified using the circular RNA immunoprecipitation method and the luciferase reporter assay. The interaction between SERPINH1 and transcription factor c-Myc was verified through Co-immunoprecipitation (Co-IP) and immunofluorescence. The effect of c-Myc on the generation of circCAMSAP1 was examined through RT-qPCR and chromatin immunoprecipitation. Finally, the splicing factors that promote the production of circCAMSAP1 were explored by RT-qPCR and RNA immunoprecipitation (RIP). Results We found that circCAMSAP1 was highly expressed in NPC tissues and promoted NPC proliferation and metastasis. Additionally, circCAMSAP1 promoted SERPINH1 expression through improved SERPINH1 mRNA stability by binding to the 3′-untranslated region (3’UTR) of SERPINH1. Highly expressed SERPINH1 reduced the ubiquitination-degradation rate of c-Myc, causing increased tumorigenesis. Meanwhile, c-Myc, cooperating with splicing factor 10 (SRSF10), could also promote CAMSAP1 pre-mRNA transcription and back-splicing, forming a positive feedback of circCAMSAP1 production, resulting in the proliferation and metastasis of NPC. Conclusions Our findings revealed that circCAMSAP1 promotes NPC proliferation and metastasis by binding to the 3’UTR of SERPINH1, suggesting that the positive feedback of circCAMSAP1-SERPINH1-c-Myc may serve as a prognostic biomarker or therapeutic target in patients with NPC.

Background: No effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination. Methods: Control of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC. Findings: We found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8+ T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8+ T cells, avasimibe significantly increased the number of tumor-infiltrating CD8+ T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells. Interpretation: Avasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer. Keywords: Chemoimmunoprevention, Kras, Peptide vaccine, Avasimibe, Cholesterol metabolism, T cell

Bexarotene has shown inhibition of lung and mammary gland tumorigenesis in preclinical models and in clinical trials. The main side effects of orally administered bexarotene are hypertriglyceridemia and hypercholesterolemia. We previously demonstrated that aerosolized bexarotene administered by nasal inhalation has potent chemopreventive activity in a lung adenoma preclinical model without causing hypertriglyceridemia. To facilitate its future clinical translation, we modified the formula of the aerosolized bexarotene with a clinically relevant solvent system. This optimized aerosolized bexarotene formulation was tested against lung squamous cell carcinoma mouse model and lung adenocarcinoma mouse model and showed significant chemopreventive effect. This new formula did not cause visible signs of toxicity and did not increase plasma triglycerides or cholesterol. This aerosolized bexarotene was evenly distributed to the mouse lung parenchyma, and it modulated the microenvironment in vivo by increasing the tumor-infiltrating T cell population. RNA sequencing of the lung cancer cell lines demonstrated that multiple pathways are altered by bexarotene. For the first time, these studies demonstrate a new, clinically relevant aerosolized bexarotene formulation that exhibits preventive efficacy against the major subtypes of lung cancer. This approach could be a major advancement in lung cancer prevention for high risk populations, including former and present smokers.

Background Circular RNAs (circRNAs) are widely expressed in human cells and are closely associated with cancer development. However, they have rarely been investigated in the context of nasopharyngeal carcinoma (NPC). Methods We screened a new circRNA, circRNF13, in NPC cells using next-generation sequencing of mRNA. Reverse transcription polymerase chain reaction and RNA fluorescence in situ hybridization were used to detect circRNF13 expression in 12 non-tumor nasopharyngeal epithelial (NPE) tissues and 36 NPC samples. Cell proliferation was detected using MTT and flow cytometry assays, and colony formation capability was detected using colony formation assays. Cell migration and invasion were analyzed using wound-healing and Transwell assays, respectively. Cell glycolysis was analyzed using the Seahorse glycolytic stress test. Glucose transporter type 1 (GLUT1) ubiquitination and SUMOylation modifications were analyzed using co-immunoprecipitation and western blotting. CircRNF13 and Small Ubiquitin-like Modifier 2 (SUMO2) interactions were analyzed using RNA pull-down and luciferase reporter assays. Finally, to test whether circRNF13 inhibited NPC proliferation and metastasis in vivo, we used a xenograft nude mouse model generated by means of subcutaneous or tail vein injection. Results We found that circRNF13 was stably expressed at low levels in NPC clinical tissues and NPC cells. In vitro and in vivo experiments showed that circRNF13 inhibited NPC proliferation and metastasis. Moreover, circRNF13 activated the SUMO2 protein by binding to the 3′- Untranslated Region (3′-UTR) of the SUMO2 gene and prolonging the half-life of SUMO2 mRNA. Upregulation of SUMO2 promotes GLUT1 degradation through SUMOylation and ubiquitination of GLUT1, which regulates the AMPK-mTOR pathway by inhibiting glycolysis, ultimately resulting in the proliferation and metastasis of NPC. Conclusions Our results revealed that a novel circRNF13 plays an important role in the development of NPC through the circRNF13-SUMO2-GLUT1 axis. This study implies that circRNF13 mediates glycolysis in NPC by binding to SUMO2 and provides an important theoretical basis for further elucidating the pathogenesis of NPC and targeted therapy.

Epstein–Barr virus (EBV) infection is an established cause of nasopharyngeal carcinoma (NPC) and is involved in a variety of malignant phenotypes, including tumor immune escape. EBV can encode a variety of circular RNAs (circRNA), however, little is known regarding the biological functions of these circRNAs in NPC. In this study, EBV-encoded circBART2.2 was found to be highly expressed in NPC where it upregulated PD-L1 expression and inhibited T-cell function in vitro and in vivo. circBART2.2 promoted transcription of PD-L1 by binding the helicase domain of RIG-I and activating transcription factors IRF3 and NF-κB, resulting in tumor immune escape. These results elucidate the biological function of circBART2.2, explain a novel mechanism of immune escape caused by EBV infection, and provide a new immunotherapy target for treating NPC. Significance: This work demonstrates that circBART2.2 binding to RIG-I is essential for the regulation of PD-L1 and subsequent immune escape in nasopharyngeal carcinoma.

Identifying factors underlying resistance to immune checkpoint therapy (ICT) is still challenging. Most cancer patients do not respond to ICT and the availability of the predictive biomarkers is limited. Here, we re-analyze a publicly available single-cell RNA sequencing (scRNA-seq) dataset of melanoma samples of patients subjected to ICT and identify a subset of macrophages overexpressing TREM2 and a subset of gammadelta T cells that are both overrepresented in the non-responding tumors. In addition, the percentage of a B cell subset is significantly lower in the non-responders. The presence of these immune cell subtypes is corroborated in other publicly available scRNA-seq datasets. The analyses of bulk RNA-seq datasets of the melanoma samples identify and validate a signature - ImmuneCells.Sig - enriched with the genes characteristic of the above immune cell subsets to predict response to immunotherapy. ImmuneCells.Sig could represent a valuable tool for clinical decision making in patients receiving immunotherapy., Most cancer patients do not respond to immune checkpoint therapies and the availability of predictive biomarkers is limited. Here the authors propose a signature enriched for genes of TREM2hi macrophages and γδ T cells to predict response to immunotherapy.

Background: Red ginseng has long been used as a traditional medicine for a variety of maladies. Ginsenosides are the active components of ginseng but are limited by their low oral bioavailability. Methods: We evaluated several types of red ginseng extracts for their ability to inhibit lung tumor formation and growth induced by the carcinogen benzo(a)pyrene [B(a)P] in A/J mice. The concentrations of various ginsenosides were quantified in these ginseng extracts using the methods of ultra-performance liquid chromatography tandem mass spectrometry analysis to identify the ginsenosides that may contribute to cancer prevention. We next explored whether inhibition of P-glycoprotein by verapamil could increase the oral bioavailability of ginsenoside by using CaCo-2 cell transcellular transport and in situ mouse intestinal perfusion models. The plasma and intestine concentration of ginsenoside and cancer preventive effect of ginsenoside combined with verapamil in A/J mice were also detected by using B(a)P-induced mouse cancer model.Results: We found that treatment with one type of red ginseng (Korean red ginseng B, KRGB) led to a significant reduction of tumor load compared with other types of red ginseng. KRGB contained the highest concentration of ginsenoside Rg3 among these red ginseng extracts, suggested that Rg3 may play an important role in its preventive efficacy. Our study showed that Rg3 had a relatively poor bioavailability, and co-administration of verapamil decreased the efflux ratio of Rg3 in Caco-2 cells, increased the absorption rate of Rg3 in rat small intestine, increased the plasma and intestine concentration of Rg3 in A/J mice, and enhanced the cancer preventive effect of Rg3 against B(a)P induced lung tumorigenesis. Conclusions: Ginsenosides Rg3 is one of the key components of read ginseng that may be responsible for its efficacy against lung carcinogenesis in mice. Rg3 appears to be the substrate of P-glycoprotein, and inhibition of P-glycoprotein could enhance its oral bioavailability.

Expressed on cells of the myeloid and lymphoid lineages, V-domain Ig Suppressor of T cell Activation (VISTA) is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. Using a tripeptide small molecule antagonist of VISTA CA170, we found that it exhibited potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. Remarkably, lung tumor development was almost completely suppressed when CA170 was combined with an MHCII-directed KRAS peptide vaccine. Flow cytometry and single-cell RNA sequencing (scRNA-seq) revealed that CA170 increased CD8+ T cell infiltration and enhanced their effector functions by decreasing the tumor infiltration of myeloid-derived suppressor cells (MDSCs) and Regulatory T (Treg) cells, while the Kras vaccine primarily induced expansion of CD4+ effector T cells. VISTA antagonism by CA170 revealed strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory and regulatory T cells, and drives an adaptive T cell tumor-specific immune response that enhances the efficacy of the KRAS vaccine., Pan, Chen, Zhang et al. investigated the anti-cancer efficacy of CA170, a small molecule agonist of VISTA, on mouse lung tumorigenesis. When combined with a MHCII-directed KRAS peptide vaccine, the authors reported that CA170 suppressed lung tumor development by increasing CD8+ T cell and decreasing MDSC and Treg infiltration, while the Kras vaccine induced CD4+ effector T cell expansion

Hydroxy genkwanin (HGK), a flavonoid compound from natural resources, showed good inhibition against the growth of breast tumor cells. However, the poor solubility restricted the further study and the in vivo drug delivery of HGK. We prepared HGK nanosuspensions by antisolvent precipitation method and investigated their characterization, stability, hemolysis probability, release behavior in vitro, antitumor activity in vitro and in vivo, and preliminary safety through acute toxicity experiments. The resultant HGK nanosuspensions (HGK-NSps) showed an average diameter of (261.1 ± 4.8 nm), a narrow particle size distribution (PDI of 0.12 ± 0.01), spherical morphology, high drug-loading content (39.9 ± 2.3%, w/w), and good stability in various physiological media. HGK-NSps was safe for intravenous injection at low concentration and HGK was slowly released from the obtained nanosuspensions. HGK-NSps showed stronger cytotoxicity than free HGK against many tumor cells in vitro. Especially against MCF-7 cells, the IC50 value was decreased to 1.0 μg/mL, 5-fold lower than the HGK solution. In the in vivo antitumor activity study HGK-NSps (40 mg/kg) displayed a similar therapeutic effect to that of the paclitaxel injection (8 mg/kg). The preliminary acute toxicity test showed that even at the highest dose of 360 mg/kg (iv), HGK-NSps had 100% of mice survival and all the mice were in a good state, suggesting a maximum tolerated dose more than 360 mg/kg. The effective antitumor effect and good tolerance showed HGK-NSps were likely to become a safe and effective antitumor drug for the treatment of breast cancer in the future.

Summary Although PD-1-blocking immunotherapies demonstrate significant therapeutic promise, a subset of the patients could develop hyperprogressive disease (HPD) with accelerated tumor growth after anti-PD1 immunotherapy. To elucidate the underlying mechanisms, we compared the mutational and transcriptional landscapes between the pre- and post-therapy tumors of two patients developing HPD after anti-PD-1 immunotherapy. In post-therapy HPD tumors, somatic mutations were found in known cancer genes, including tumor suppressor genes such as TSC2 and VHL, along with transcriptional upregulation of oncogenic pathways, including IGF-1, ERK/MAPK, PI3K/AKT, and TGF-β. We found that post-therapy HPD tumors were less immunogenic than pre-therapy tumors, concurrent with an increased presence of ILC3 cells, a subset of innate lymphoid cells. We also developed a gene expression signature predictive of HPD. In summary, we identified the genomics and immune features associated with HPD, which may help identify patients at risk of adverse clinical outcome after anti-PD-1 immunotherapy., Graphical Abstract, Highlights • Mutations/expression changes occur in hyperprogressive tumors after anti-PD-1 therapy • Immune cell population abundance pattern changed in the hyperprogressive tumors • ILC3 cells may be enriched in the hyperprogressive tumors after anti-PD-1 therapy • Post-therapy hyperprogressive tumors were less immunogenic than pre-therapy tumors, Physiology (170590663/189723279); Immunology (186131996Physiology; Biotechnology; Cell Biology; Omics

Summary: We synthesized a mitochondria-targeted honokiol (Mito-HNK) that facilitates its mitochondrial accumulation; this dramatically increases its potency and efficacy against highly metastatic lung cancer lines in vitro, and in orthotopic lung tumor xenografts and brain metastases in vivo. Mito-HNK is >100-fold more potent than HNK in inhibiting cell proliferation, inhibiting mitochondrial complex ǀ, stimulating reactive oxygen species generation, oxidizing mitochondrial peroxiredoxin-3, and suppressing the phosphorylation of mitoSTAT3. Within lung cancer brain metastases in mice, Mito-HNK induced the mediators of cell death and decreased the pathways that support invasion and proliferation. In contrast, in the non-malignant stroma, Mito-HNK suppressed pathways that support metastatic lesions, including those involved in inflammation and angiogenesis. Mito-HNK showed no toxicity and targets the metabolic vulnerabilities of primary and metastatic lung cancers. Its pronounced anti-invasive and anti-metastatic effects in the brain are particularly intriguing given the paucity of treatment options for such patients either alone or in combination with standard chemotherapeutics. : Natural Product Chemistry; Immunology; Medicinal and Aromatic Plants Subject Areas: Natural Product Chemistry, Immunology, Medicinal and Aromatic Plants

Lung squamous cell carcinoma (LUSC) is a major subtype of Non-Small Cell Lung Cancer. To increase our understanding of the LUSC pathobiology, we performed exome sequencing and RNA-seq in 16 murine carcinogen-induced LUSC tumors and 8 normal murine lung tissue samples. Additionally, we conducted single-cell RNA-seq on two independent tumors from the same murine model. We identified a list of 59 cancer genes recurrently mutated in the mice LUSC tumors, 47 (80%) of which were also mutated in human LUSCs. At the single cell level, we detected unique clonal mutation patterns for each of the two LUSC tumors, being initiated from clones carrying the mutant Igfbp7 and Trp53 genes, respectively. We also identified an expression signature serving as an effective classifier for LUSC tumors and a strong predictor of survival outcomes of lung cancer patients. Lastly, we found that some of the mutant LUSC genes were associated with the significantly altered tumoral expression of inhibitory immune checkpoint genes such as PD-L1, VISTA, TIM3 and LAG3 in human LUSCs. The novel findings of clonal evolution, mutational landscapes and expression signatures of LUSC suggested new targets for the overall LUSC therapy and the immunotherapy of LUSC.

Lung cancer is the leading cause of mortality worldwide. MicroRNAs (miRNAs) are potential candidates for lung cancer therapy. However, a major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting exposure to healthy cells. In our previous studies on aerosolized let-7b in lung cancer prevention, let-7b showed good inhibition of B[a]P-induced lung adenoma with no side effects. In this study, we found that aerosolized let-7b decreased tumor growth in the LKR13 (KRAS mutant) syngeneic mouse model. Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor immune microenvironment, suggesting that let-7 microRNAs may promote antitumor immunity in vivo. Single cell RNA sequencing (scRNAseq) data showed that let-7b treatment decreased the expression of PD-1 in CD8+ T cells and reduced PD-L1 expression in lung tumor cells. Let-7b treatment also significantly changed the percentages of distinct CD8+ tumor-infiltrating lymphocytes (TIL) states. The proportion of CD8+ T cells mediating anti-tumor functions (EM-like CD8+ TILs) was increased significantly by let-7b treatment compared to control. In contrast, the CD8+ T cell subpopulation that has negative effects on anti-tumor immune response, exhausted CD8+ TILs, was significantly decreased by let-7b. Flow cytometry data showed that Let-7b treatment led to the accumulation of CD8+ T cells, granzyme B+ CD8+ T cells and IFN-γ+ CD8+ T cells in tumors, and a decrease of intratumoral Granulocyte-like myeloid derived suppressor cells (G-MDSC) cells. Our results suggest that the in vivo tumor-suppressive efficacy of let-7 is mediated, at least in part, by immune-promoting effects via down-regulating PD-L1 in tumors and/or PD-1 on CD8+ T cells. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment [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 2369.

Lung cancer is the leading cause of cancer mortality worldwide. More than 85% of lung cancers are non-small cell lung cancer (NSCLC). EGFR mutations occur in 47.9% of Asia-Pacific patients with NSCLC and 19.2% of Western patients. The most common EGFR mutations (>90%) are deletions in exon 19 and/or point mutations in exon 21 (L858R). Th1 helper cellular immunity is critical for immunotherapy-mediated tumor regression. We have previously characterized an MHC-II-restricted EGFR multi-peptide vaccine (two peptides: “SCVRACGADSYEMEEDGVRK” and “VWSYGVTVWELMTFGSKPY”) (EGFR-V) that targets the EGFR protein and decreases EGFR-driven lung tumorigenesis by ~80% in EGFRL858R transgenic mice that were vaccinated before doxycycline-induction of the EGFR protein [1]. However, diminished efficacy was observed when this MHC-II-restricted EGFR multi-peptide vaccine was given two weeks after doxycycline induction of the EGFR protein, suggesting that expression of the EGFR oncoprotein significantly increased the immunosuppressive microenvironment. JHU083, an orally bioavailable glutamine antagonist, has recently been shown to not only inhibit tumor growth but also boost anti-cancer immunity [2]. To determine if JHU083 could potentiate the efficacy of EGFR vaccine in a post-initiation setting, we evaluated in vivo antitumor efficacy of EGFR vaccine combined with JHU083 using an EGFRL858R transgenic mouse model. JHU083 inhibited tumor burden by 31% and EGFR vaccine suppressed tumor burden by 33%, whereas combining JHU083 with anti-EGFR peptide vaccine had an additive effect (54% tumor inhibition). Mechanistically, JHU083 by itself, markedly reduced the immunosuppressive monocytic myeloid-derived suppressor cells (MDSCs) in lung tissues. The combination of JHU083 and the vaccine significantly reduced Tregs in lung tissues. The anti-EGFR vaccine primarily induced expansion of antigen specific antitumor CD4+ effector T cells. Long term administration of JHU-083 did not decrease bodyweight in mice. Together with previous results, these data suggest that JHU083 could be used to reshape the tumor microenvironment toward one that enhances antitumor T cell responses and could further enhance the efficacy of the EGFR anticancer vaccine. References1.Ebben, J.D., et al., Epidermal growth factor receptor derived peptide vaccination to prevent lung adenocarcinoma formation: An in vivo study in a murine model of EGFR mutant lung cancer. Mol Carcinog, 2016. 55(11): p. 1517-1525.2.Leone, R.D., et al., Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science, 2019. 366(6468): p. 1013-1021. Citation Format: Mofei Huang, Jing Pan, Qi Zhang, Shizuko Sei, Robert Shoemaker, Ronald Lubet, Yian Wang, Barbara Slusher, Ming You. Potentiation of EGFR peptide cancer vaccine by an orally bioavailable glutamine antagonist prodrug JHU-083 [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 440.

Immunotherapy using monoclonal antibodies to block immune checkpoints is becoming a mainstream therapy for multiple late-stage cancers owing to their efficacy in improving clinical outcomes. However, relatively low response rates and persistent clinical side effects indicate the need for additional strategies to block immune-suppressive pathways. VISTA (V-domain Ig Suppressor of T cell Activation), which is expressed on cells of the myeloid and lymphoid lineages, is an emerging target for cancer immunotherapy. Blocking VISTA activates both innate and adaptive immunity to eradicate tumors in mice. We found that CA170, a novel orally bioavailable tripeptide small molecule antagonist of VISTA and PD-L1/PD-L2, has potent anticancer efficacy on carcinogen-induced mouse lung tumorigenesis. Remarkably, lung tumor development was almost completely suppressed when CA170 was combined with an MHC-II-directed KRAS peptide vaccine. Flow cytometry and single cell RNA sequencing (scRNA-seq) revealed that CA170 increases CD8+ T cell infiltration and effector functions by decreasing the tumor infiltration of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs), while the KRAS vaccine primarily induces expansion of CD4+ effector T cells. Inhibition of VISTA by CA170 has strong efficacy against lung tumorigenesis with broad immunoregulatory functions that influence effector, memory and regulatory T cells, and promotes an adaptive T cell tumor-specific immune response that enhances efficacy of the KRAS vaccine. Citation Format: Jing Pan, Yao Chen, Qi Zhang, Achia Khatun, Katie Palen, Li Wang, Chuanjia Yang, Bryon D. Johnson, Charles R. Myers, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Weiguo Cui, Ming You. Inhibition of lung tumorigenesis by a novel small molecule CA170 targeting the immune checkpoint protein VISTA [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 1618.

Lung cancer is the leading cause of cancer death in the United States. Metastasis to lymph nodes and distal organs, especially brain, leads to severe complications and death. Preventing lung cancer development and metastases is an important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in the extracts of magnolia bark, has a favorable bioavailability profile and recently has been shown to readily cross the blood–brain barrier. In the current study, we evaluated the antimetastatic effects of HNK in both the lymph node and brain mouse models of lung tumor metastasis. We tested the efficacy of HNK in preventing 18 H2030-BrM3 cell (brain-seeking human lung tumor cells) migration to lymph node or brain. In an orthotopic mouse model, HNK significantly decreased lung tumor growth compared with the vehicle control group. HNK also significantly reduced the incidence of lymph node metastasis and the weight of mediastinal lymph nodes. In a brain metastasis model, HNK inhibits metastasis of lung cancer cells to the brain to approximately one third of that observed in control mice. We analyzed HNK's mechanism of action, which indicated that its effect is mediated primarily by inhibiting the STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation irrespective of the mutation status of EGFR, and knockdown of STAT3 abrogated both the antiproliferative and the antimetastatic effects of HNK. These observations suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer metastasis. Cancer Prev Res; 10(2); 133–41. ©2016 AACR.

Immune checkpoint blockade therapy (ICBT) can unleash T-cell responses against cancer. However, only a small fraction of patients exhibited responses to ICBT. The role of immune checkpoints in cancer cells is not well understood. In this study, we analyzed T-cell coinhibitory/costimulatory genes across more than 1100 samples of the Cancer Cell Line Encyclopedia (CCLE). Nearly 90% of such genes were not expressed or had low expression across the CCLE cancer cell lines. Cell line screening showed the enrichment of cancer cells deprived of the expression of CD27, CEACAM1, CTLA4, LRIG1, PDCD1LG2, or TNFRSF18, suggesting their role as tumor suppressor. The metagene expression signature derived from these six genes - Immu6Metagene was associated with prolonged survival phenotypes. A common set of five oncogenic pathways were significantly inhibited in different types of tumors of the cancer patients with good survival outcome and high Immu6Metagene signature expression. These pathways were TGF-β signaling, angiogenesis, EMT, hypoxia and mitotic process. Our study showed that oncoimmunology related molecules especially the six genes of the Immu6Metagene signature may play the tumor suppressor role in certain cancers. Therefore, the ICBT targeting them should be considered in such context to improve the efficacy.

Genetic instability of tumor cells often leads to the occurrence of a large number of mutations, and expression of non-synonymous mutations can produce tumor-specific antigens called neoantigens. Neoantigens are highly immunogenic as they are not expressed in normal tissues. They can activate CD4+ and CD8+ T cells to generate immune response and have the potential to become new targets of tumor immunotherapy. The development of bioinformatics technology has accelerated the identification of neoantigens. The combination of different algorithms to identify and predict the affinity of neoantigens to major histocompatibility complexes (MHCs) or the immunogenicity of neoantigens is mainly based on the whole-exome sequencing technology. Tumor vaccines targeting neoantigens mainly include nucleic acid, dendritic cell (DC)-based, tumor cell, and synthetic long peptide (SLP) vaccines. The combination with immune checkpoint inhibition therapy or radiotherapy and chemotherapy might achieve better therapeutic effects. Currently, several clinical trials have demonstrated the safety and efficacy of these vaccines. Further development of sequencing technologies and bioinformatics algorithms, as well as an improvement in our understanding of the mechanisms underlying tumor development, will expand the application of neoantigen vaccines in the future.

Background: Chemopreventive agent (CPA) treatment is one of the main preventive options for lung cancer. However, few studies have been done on pharmacodynamic biomarkers of known CPAs for lung cancer. Materials and methods: In this study, we treated mouse models of lung squamous cell carcinoma with three different CPAs (MEK inhibitor: AZD6244, PI-3K inhibitor: XL-147 and glucocorticoid: Budesonide) and examined circulating exosomal miRNAs in the plasma of each mouse before and after treatment. Results: Compared to baselines, we found differentially expressed exosomal miRNAs after AZD6244 treatment (n = 8, FDR &lt, 0.05, n = 55, raw p-values &lt, 0.05), after XL-147 treatment (n = 4, FDR &lt, n = 26, raw p-values &lt, 0.05) and after Budesonide treatment (n = 1, FDR &lt, n = 36, raw p-values &lt, 0.05). In co-expression analysis, we found that modules of exosomal miRNAs reacted to CPA treatments differently. By variable selection, we identified 11, 9 and nine exosomal miRNAs as predictors for AZD6244, XL-147 and Budesonide treatment, respectively. Integrating all the results, we highlighted 4 miRNAs (mmu-miR-215-5p, mmu-miR-204-5p, mmu-miR-708-3p and mmu-miR-1298-5p) as the key for AZD6244 treatment, mmu-miR-23a-3p as key for XL-147 treatment, and mmu-miR-125a-5p and mmu-miR-16-5p as key for Budesonide treatment. Conclusions: This is the first study to use circulating exosomal miRNAs as pharmacodynamic biomarkers for CPA treatment in lung cancer.

The immune system plays important roles in tumor development. According to the immune-editing theory, immune escape is the key to tumor survival, and exploring the mechanisms of tumor immune escape can provide a new basis for the treatment of tumors. In this review, we describe the mechanisms of natural killer group 2D (NKG2D) receptor and NKG2D ligand (NKG2DL) in tumor immune responses. Natural killer (NK) cells are important cytotoxic cells in the immune system, and the activated NKG2D receptor on the NK cell surface can bind to NKG2DL expressed in tumor cells, enabling NK cells to activate and kill tumor cells. However, tumors can escape the immune clearance mediated by NKG2D receptor/NKG2DL through various mechanisms. The expression of NKG2D receptor on NK cells can be regulated by cells, molecules, and hypoxia in the tumor microenvironment. Tumor cells regulate the expression of NKG2DL at the level of transcription, translation, and post-translation and thereby escape recognition by NK cells. In particular, viruses and hormones have special mechanisms to affect the expression of NKG2D receptor and NKG2DL. Therefore, NKG2D\NKG2DL may have applications as targets for more effective antitumor therapy.

The delivery of microRNA (miRNA) via inhalation is a potential strategy for lung cancer prevention in high risk individuals such as current and former smokers. Previous studies have shown that intratracheal or intranasal exposure of genetically engineered mice to viral vectors expressing let-7 miRNA resulted in a reduced lung tumor burden. However, these studies were done in genetically engineered mouse models that developed highly aggressive tumors more relevant for a treatment regimen. In this study, we investigated the efficacy of let-7b in lung cancer prevention. An aerosolized let-7b miRNA formulation was tested in the benzo(a)pyrene (B[a]P) mouse model. The particle size of the let-7b miRNA aerosol has been systematically characterized as particles with sizes between 1.9 to 35 nm (average size of 29.0 nm). Following the aerosol formulation evaluation, the biodistribution of let-7b in the lung by tail vein injection and aerosolized delivery were compared. The distribution of the aerosolized let-7b produced higher levels of the miRNA in the lung compared with systematic delivery. The aerosol delivery of let-7b had no effect on animal body weight nor were there any other signs of toxicity. The development of an aerosolized let-7b formulation inhibited B[a]P - induced lung adenoma volume by 72% in A/J mice. Collectively, these findings show that aerosolized miRNA is a promising approach for lung cancer prevention. Citation Format: Qi Zhang, Jing Pan, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Chemoprevention of aerosolized Let-7b in mouse lung cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2540.

Kirsten rat sarcoma viral oncogene homolog (KRAS) gene encodes a small GTPase that cycles between GDP and GTP-bound states, which modulates important cell surface receptors essential for cell proliferation and survival. It is one of the most common drivers for many types of cancers, is present in up to 25% all human tumors, and 80% of mutation occurs in codon12. Efforts to target KRAS-driven cancers preventively or therapeutically have been unsuccessful. We recently formulated a multi-peptide KRAS vaccine (KVAX), which targets both wild-type and G12D mutant forms of KRAS and has 100% sequence homology between human and mouse. KVAX elicited strong immunologic response and exhibited striking cancer preventive efficacy when administered prior to KRAS mutation induction. However, clinical high-risk individuals may already express mutant KRAS, which has known to promote an immunosuppressive environment. Therefore, we combined KVAX with avasimibe (AVA), an ACAT1 inhibitor that modulates lipid metabolism in T cells and facilitates better TCR/peptide/MHC interactions, in syngeneic lung cancer mouse model and genetically engineered mouse model, in which mutant KRAS initiated lung tumorigenesis before vaccination. We found that the combination significantly decreased the presence of regulatory T cells in the tumor microenvironment, facilitated CD8+ T cell infiltration in tumor sites, and ultimately led to enhanced anti-cancer efficacy. Using 10X Genomics, we performed single cell RNA-seq on lung tumors from mice treated with KVAX and AVA and found significant increases in APC monocytes and CD4+ Th1 cells, especially increases in the effector/memory T cell population. Meanwhile, KVAX also decreased Tregs and M2 macrophages. These studies include the evaluation of tumor-infiltrating immune cells by single-cell RNA-seq (scRNA-seq) and provide a more detailed mechanisms of action of KVAX alone or in combination with AVA in modulating the tumor immune microenvironment. Citation Format: Jing Pan, Qi Zhang, Katie Palen, Bryon Johnson, Li Wang, Shizuko Sei, Robert H. Shoemaker, Ronald A. Lubet, Yian Wang, Ming You. A novel combination of a multipeptide KRAS vaccine and an ACAT1 inhibitor to prevent lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4584.

Triple negative breast cancer (TNBC) is the most aggressive sub-type of breast cancer, comprising approximately 20% of all breast cancers, and has a poor prognosis due to its aggressive behavior and lack of effective targeted therapies. In the present study, we employed transcriptome and protein analyses to select genes that are uniquely overexpressed in human triple-negative breast cancer. Overexpression of selected genes, Cyclin A2, CDK1, Cyclin E2, PRC1, Kif15, TPX2, Ki67, Top2A, were confirmed on human breast cancer tissue microarrays and in TNBC derived cell lines from C3(1)/TAg mice. Antigen-specific MHC class II epitopes with promiscuous high-binding affinity were identified using the “combined scoring system”, based on their potential to elicit a selective Th1 immunity. Furthermore, all peptides were selected to have over 85% amino acid sequence homology between human and mice for potential clinical translation. Strong immunogenic peptides from five antigens were selected by immunogenicity testing in tumor naïve mice based on high IFN-γ and low IL-10 ELISpot response data. The selected peptides, Cyclin A2 (#335), CDK1 (#113), Cyclin E2 (#93, 298, 334), KIF15 (#129), and Top2A (#236, 410, 606), were combined to form a multi-antigen, multi-peptide vaccine (MAMPV). The tumor preventive efficacy of the MAMPV was evaluated in syngraft study using a TNBC model: C3(1)/TAg-REAR mice and a TNBC cell line derived from C3(1)/TAg (M6). MAMPV-vaccinated animals demonstrated a significant reduction in tumor volume, and significantly higher tumor infiltration of CD4+ T cells compared to the control animals. Pathological examination of major organs did not reveal any toxicity associated with the vaccine. Similar tumor preventive efficacy was demonstrated in genetically engineered mouse model (GEMM) using C3(1)/TAg mice. Taken together, our data indicate that formulation of the multi-antigen multi-peptide vaccine is highly immunogenic and has the potential to be safe and efficacious in the immunoprevention of TNBC. Citation Format: Yian Wang. Immunoprevention of triple negative breast cancer by a multi-antigen, multi-peptide vaccine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4563.

Recent studies have shown that on one hand, tumors need to obtain a sufficient energy supply, and on the other hand they must evade the body’s immune surveillance. Because of their metabolic reprogramming characteristics, tumors can modify the physicochemical properties of the microenvironment, which in turn affects the biological characteristics of the cells infiltrating them. Regulatory T cells (Tregs) are a subset of T cells that regulate immune responses in the body. They exist in large quantities in the tumor microenvironment and exert immunosuppressive effects. The main effect of tumor microenvironment on Tregs is to promote their differentiation, proliferation, secretion of immunosuppressive factors, and chemotactic recruitment to play a role in immunosuppression in tumor tissues. This review focuses on cell metabolism reprogramming and the most significant features of the tumor microenvironment relative to the functional effects on Tregs, highlighting our understanding of the mechanisms of tumor immune evasion and providing new directions for tumor immunotherapy.

Nasopharyngeal carcinoma (NPC) is a unique malignant cancer with high metastasis. Because the early symptoms of NPC patients are not obvious, most patients have distant metastases when diagnosed, which makes treatment difficult. Long non-coding RNAs (lncRNAs) are emerging as important regulators in human carcinogenesis. LncRNAs have been increasingly identified but remain largely unknown in NPC. Therefore, we performed gene expression profiling to screen for altered expression of lncRNAs in NPC tissues and adjacent samples. One lncRNA, LOC284454, was upregulated and associated with poor prognosis in NPC. In in vivo and in vitro assays, LOC284454 promoted the migration and invasion capacity of NPC cells. Mass spectrometry combined with bioinformatics suggested that LOC284454 affected the cytoskeletal and adhesion-related Rho/Rac signaling pathways. LOC284454 may be a potential novel treatment target and is expected to be a new diagnostic and prognostic marker in patients with NPC.

As a consequence of exposure to environmental toxicants, a “field cancerization” effect occurs in the lung, resulting in the development of a field of initiated, but morphologically normal appearing cells within a damaged epithelium containing mutations in oncogene or tumor suppressor genes. Unlike humans, whose airway field of injury associated with lung cancer has long been investigated with airway brushings obtained via bronchoscopy, no methods are available for similar studies in the mouse due to the small size of the murine airways. In this protocol, we describe a detailed method for performing airway brushing from a live mouse, which enables repeated sampling from the same mouse and thus, mimicking the bronchoscopy protocol used in humans. Using this approach in the N-nitroso-tris-chloroethylurea (NTCU)-induced mouse lung squamous cell carcinoma (SCC) model, we isolated airway epithelial cells with intact cell membrane structure and then performed transcriptome sequencing (RNA-Seq). We found activation of the PI3K signaling network to be the most significant in cytologically normal bronchial airway epithelial cells of mice with preneoplastic lung SCC lesions. Prolonged exposure to NTCU also induced activation of NF-kappaB (NFƙB), the downstream pathway of PI3K; this NTCU-induced lung SCC progression can be reversed by blocking the NFƙB pathway.

Non-small cell lung cancer (NSCLC) patients with an epidermal growth factor receptor (EGFR) mutation have benefited from treatment of reversible EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Acquisition of a secondary mutation in EGFR T790M is the most common mechanism of resistance to first generation EGFR TKIs, resulting in therapeutic failure. Afatinib is a second generation of EGFR TKI that showed great efficacy against tumors bearing the EGFR T790M mutation, but it failed to show the improvement on overall survival of lung cancer patients with EGFR mutations possibly because of novel acquired resistance mechanisms. Currently, there are no therapeutic options available for lung cancer patients who develop acquired resistance to afatinib. To identify novel resistance mechanism(s) to afatinib, we developed afatinib resistant cell lines from a parental human-derived NSCLC cell line, H1975, harboring both EGFR L858R and T790M mutations. We found that activation of the insulin-like growth factor 1 receptor (IGF1R) signaling pathway contributes to afatinib resistance in NSCLC cells harboring the T790M mutation. IGF1R knockdown not only significantly sensitizes resistant cells to afatinib, but also induces apoptosis in afatinib resistance cells. In addition, combination treatment with afatinib and linsitinib shows more than additive effects on tumor growth in in vivo H1975 xenograft. Therefore, these finding suggest that IGF1R inhibition or combination of EGFR-IGF1R inhibition strategies would be potential ways to prevent or potentiate the effects of current therapeutic options to lung cancer patients demonstrating resistance to either first or second generation EGFR TKIs © 2015 Wiley Periodicals, Inc.

3-Bromopyruvate (3-BrPA) is an alkylating agent and a well-known inhibitor of energy metabolism. Rapamycin is an inhibitor of the serine/threonine protein kinase mTOR. Both 3-BrPA and rapamycin show chemopreventive efficacy in mouse models of lung cancer. Aerosol delivery of therapeutic drugs for lung cancer has been reported to be an effective route of delivery with little systemic distribution in humans. In this study, 3-BrPA and rapamycin were evaluated in combination for their preventive effects against lung cancer in mice by aerosol treatment, revealing a synergistic ability as measured by tumor multiplicity and tumor load compared treatment with either single-agent alone. No evidence of liver toxicity was detected by monitoring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. To understand the mechanism in vitro experiments were performed using human non–small cell lung cancer (NSCLC) cell lines. 3-BrPA and rapamycin also synergistically inhibited cell proliferation. Rapamycin alone blocked the mTOR signaling pathway, whereas 3-BrPA did not potentiate this effect. Given the known role of 3-BrPA as an inhibitor of glycolysis, we investigated mitochondrial bioenergetics changes in vitro in 3-BrPA–treated NSCLC cells. 3-BrPA significantly decreased glycolytic activity, which may be due to adenosine triphosphate (ATP) depletion and decreased expression of GAPDH. Our results demonstrate that rapamycin enhanced the antitumor efficacy of 3-BrPA, and that dual inhibition of mTOR signaling and glycolysis may be an effective therapeutic strategy for lung cancer chemoprevention. Cancer Prev Res; 8(4); 318–26. ©2015 AACR.

PARK2, a gene associated with Parkinson disease, is a tumor suppressor in human malignancies. Here, we show that c.823C>T (p.Arg275Trp), a germline mutation in PARK2, is present in a family with eight cases of lung cancer. The resulting amino acid change, p.Arg275Trp, is located in the highly conserved RING finger 1 domain of PARK2, which encodes an E3 ubiquitin ligase. Upon further analysis, the c.823C>T mutation was detected in three additional families affected by lung cancer. The effect size for PARK2 c.823C>T (odds ratio = 5.24) in white individuals was larger than those reported for variants from lung cancer genome-wide association studies. These data implicate this PARK2 germline mutation as a genetic susceptibility factor for lung cancer. Our results provide a rationale for further investigations of this specific mutation and gene for evaluation of the possibility of developing targeted therapies against lung cancer in individuals with PARK2 variants by compensating for the loss-of-function effect caused by the associated variation.

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared with mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 μmol/g diet or 2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials. Cancer Prev Res; 8(1); 77–85. ©2014 AACR.

Airway epithelial cells are prone to the damage caused by lung cancer risk factors, such as cigarette smoking. Little is known about surrogate biomarkers in the bronchial airway epithelium that can be used to assess the effect of potential chemoprevention drugs on lung adenocarcinoma formation/progression. Pioglitazone has been suggested as a chemoprevention drug for lung cancer. To study the mechanisms underlying the role of pioglitazone in lung cancer prevention, we performed transcriptome sequencing (RNA-Seq) and found that Kras signaling was repressed by pioglitazone treatment in the airway epithelial cells of mice with lung adenocarcinoma (FDR q = 9.8E-04). It was also found that glucose metabolic pathways were elevated in the airway epithelium of mice with lung adenocarcinomas and inhibited by pioglitazone treatment (FDR q = 0.01). Downregulation of glucose metabolism genes was also observed in lung tumors of mice treated with pioglitazone. The high-risk expression signature of elevated glucose metabolism was associated with poor survival outcome in multiple lung adenocarcinoma patient populations (P values ranging from 1.0E-9 to 5.5E-5). Our results suggest that the role of pioglitazone in preventing lung adenocarcinoma may depend on inhibiting Kras signaling and glucose metabolism, which may serve as biomarkers of agent action in the airway epithelium.

Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a nonadipogenic PPARγ agonist and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreventive efficacy of honokiol in an initiation model of lung squamous cell carcinoma (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU), which is applied topically, reliably triggering the development of SCC within 24 to 26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol-treated group (P = 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol-treated group. P = 0.004). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1–S cell-cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol changed redox status in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent. Cancer Prev Res; 7(11); 1149–59. ©2014 AACR.

Insulin-like growth factor-1 receptor (IGF-1R) is a transmembrane heterotetramer that is activated by Insulin-like growth factor 1 and is crucial for tumor transformation and survival of malignant cells. Importantly, IGF-1R overexpression has been reported in many different cancers, implicating this receptor as a potential target for anticancer therapy. Picropodophyllin (PPP) is a potent inhibitor of IGF-1R and has antitumor efficacy in several cancer types. However, the chemopreventive effect of PPP in lung tumorigenesis has not been investigated. In this study, we investigated the chemopreventive activity of PPP in a mouse lung tumor model. Benzo(a)pyrene was used to induce lung tumors, and PPP was given by nasal inhalation to female A/J mice. Lung tumorigenesis was assessed by tumor multiplicity and tumor load. PPP significantly decreased tumor multiplicity and tumor load. Tumor multiplicity and load were decreased by 52% and 78% respectively by 4 mg/ml aerosolized PPP. Pharmacokinetics analysis showed good bioavailability of PPP in lung and plasma. Treatment with PPP increased staining for cleaved caspase-3 and decreased Ki-67 in lung tumors, suggesting that the lung tumor inhibitory effects of PPP were partially through inhibition of proliferation and induction of apoptosis. In human lung cancer cell lines, PPP inhibited cell proliferation, and also inhibited phosphorylation of IGF-1R downstream targets, AKT and MAPK, ultimately resulting in increased apoptosis. PPP also reduced cell invasion in lung cancer cell lines. In view of our data, PPP merits further investigation as a promising chemopreventive agent for human lung cancer. © 2014 Wiley Periodicals, Inc.

Triple negative breast cancer (TNBC) comprises approximately 20% of all breast cancers, and it has a poor prognosis due to aggressive growth characteristics and lack of effective targeted therapies. Emerging data suggest vaccination as a promising approach to prevent TNBC as well as other cancer types, but appropriate vaccine targets need to be identified. In the present study, we employed in silico analyses to select genes that are uniquely expressed in TNBC. Topoisomerase 2 alpha (Top2A) was found to be highly expressed in human TNBC. Top2A is a key enzyme in DNA replication and has been identified as a target of anti-cancer therapy. Overexpression of Top2A was confirmed in human breast cancer tissue microarrays and in mouse TNBCs derived from C3(1)/TAg mice. Top2A-specific MHC class II epitopes with optimal binding affinity were identified using the “combined scoring system”, based on their potential to elicit a Th1 immune response. Furthermore, all peptides were selected to have 100% amino acid sequence homology between humans and mice for potential clinical translation. Four Top2A peptides with strong immunogenic responses were selected by immunization of tumor naïve mice followed by IFN-γ-based ELISPOT assays. These peptides were combined to form a multi-peptide Top2A vaccine. The anti-tumor efficacy of the Top2A vaccine was tested in a preventive setting using a syngeneic TNBC model in C3(1)/TAg-REAR mice. Vaccinated animals demonstrated a significant reduction in tumor growth when compared to the adjuvant control with tumor size decreasing from 278.1 mm3 in adjuvant control to 150.4 mm3 in vaccinated group (p < 0.05). Taken together, our data clearly demonstrate that the Top2A multi-peptide vaccine is highly immunogenic and provides immunopreventive efficacy against TNBC in mice. Citation Format: Sang Beom Lee, Jing Pan, Yueqiang Jiang, Katie Palen, Bryon Johnson, Romaine Ingrid Fernando, Shizuko Sei, Ronald Lubet, Ming You, Yian Wang. Immunoprevention of triple negative breast cancer by a TOP2A multi-peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 647.

Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects.

3-Bromopyruvate, an alkylating agent and a well-known inhibitor of energy metabolism, has been proposed as a specific anticancer agent. However, the chemopreventive effect of 3-bromopyruvate in lung tumorigenesis has not been tested. In this study, we investigated the chemopreventive activity of 3-bromopyruvate in a mouse lung tumor model. Benzo(a)pyrene was used to induce lung tumors, and 3-bromopyruvate was administered by oral gavage to female A/J mice. We found that 3-bromopyruvate significantly decreased tumor multiplicity and tumor load by 58% and 83%, respectively, at a dose of 20 mg/kg body weight by gavage. Due to the known liver toxicity of 3-bromopyruvate in animal models given large doses of 3-bromopyruvate, confirmed in this study, we decided to test the chemopreventive activity of aerosolized 3-bromopyruvate in the same lung tumor model. As expected, aerosolized 3-bromopyruvate similarly significantly decreased tumor multiplicity and tumor load by 49% and 80%, respectively, at a dose of 10 mg/mL by inhalation. Interestingly, the efficacy of aerosolized 3-bromopyruvate did not accompany any liver toxicity indicating that it is a safer route of administering this compound. Treatment with 3-bromopyruvate increased immunohistochemical staining for cleaved caspase-3, suggesting that the lung tumor inhibitory effects of 3-bromopyruvate were through induction of apoptosis. 3-Bromopyruvate also dissociated hexokinase II from mitochondria, reduced hexokinase activity, and blocked energy metabolism in cancer cells, finally triggered cancer cell death and induced apoptosis through caspase-3, and PARP in human lung cancer cell line. The ability of 3-bromopyruvate to inhibit mouse lung tumorigenesis, in part through induction of apoptosis, merits further investigation of this compound as a chemopreventive agent for human lung cancer. Cancer Prev Res; 5(5); 717–25. ©2012 AACR.

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Betanin, also called beetroot red, has been extensively used as a food colorant. In this study, the chemopreventive activity of betanin by oral consumption was investigated in two mouse lung tumor models. Vinyl carbamate (VC) and benzo(a)pyrene (B(a)P) were used to induce lung tumors, and female A/J mice were treated with betanin in drinking water. Betanin significantly decreased tumor multiplicity and tumor load induced by both carcinogens. Tumor multiplicity and tumor load were decreased by 20% and 39% in the VC lung model, and by 46% and 65% in the B(a)P lung model, respectively. Betanin reduced the number of CD31+ endothelial microvessels and increased the expression of caspase-3, suggesting that the lung tumor inhibitory effects were through induction of apoptosis and inhibition of angiogenesis. Betanin also induced apoptosis through activated caspase-3, -7, -9, and PARP in human lung cancer cell lines. Our data show that betanin significantly inhibits lung tumorigenesis in A/J mice and merits investigation as a chemopreventive agent for human lung cancer.

About 30% stage I non-small cell lung cancer (NSCLC) patients undergoing resection will recur. Robust prognostic markers are required to better manage therapy options. MicroRNAs (miRNAs) are a class of small non-coding RNAs of 19-25 nt and play important roles in gene regulation in human cancers. The purpose of this study is to identify miRNA expression profiles that would better predict prognosis of stage I NSCLC. MiRNAs extracted from 527 stage I NSCLC patients were profiled on the human miRNA expression profiling v2 panel (Illumina). The expression profiles were analyzed for their association with cancer subtypes, lung cancer brain metastasis and recurrence/relapse free survival (RFS). MiRNA expression patterns between lung adenocarcinoma and squamous cell carcinoma differed significantly with 171 miRNAs, including Let-7 family members and miR-205. Ten miRNAs associated with brain metastasis were identified including miR-145*, which inhibit cell invasion and metastasis. Two miRNA signatures that are highly predictive of RFS were identified. The first contained 34 miRNAs derived from 357 stage I NSCLC patients independent of cancer subtype, whereas the second containing 27 miRNAs was adenocarcinoma specific. Both signatures were validated using formalin-fixed paraffin embedded and/or fresh frozen tissues in independent data set with 170 stage I patients. Our findings have important prognostic or therapeutic implications for the management of stage I lung cancer patients. The identified miRNAs hold great potential as targets for histology-specific treatment or prevention and treatment of recurrent disease.

To identify the genetic determinants of colon tumorigenesis, 268 male mice from 33 inbred strains derived from different genealogies were treated with azoxymethane (AOM; 10 mg/kg) once a week for six weeks to induce colon tumors. Tumors were localized exclusively within the distal colon in each of the strains examined. Inbred mouse strains exhibit a large variability in genetic susceptibility to AOM-induced colon tumorigenesis. The mean colon tumor multiplicity ranged from 0 to 38.6 (mean = 6.5 ± 8.6) and tumor volume ranged from 0 to 706.5 mm3 (mean = 87.4 ± 181.9) at 24 weeks after the first dose of AOM. AOM-induced colon tumor phenotypes are highly heritable in inbred mice, and 68.8% and 71.3% of total phenotypic variation in colon tumor multiplicity and tumor volume, respectively, are attributable to strain-dependent genetic background. Using 97,854 single-nucleotide polymorphisms, we carried out a genome-wide association study (GWAS) of AOM-induced colon tumorigenesis and identified a novel susceptibility locus on chromosome 15 (rs32359607, P = 6.31 × 10–6). Subsequent fine mapping confirmed five (Scc3, Scc2, Scc12, Scc8, and Ccs1) of 16 linkage regions previously found to be associated with colon tumor susceptibility. These five loci were refined to less than 1 Mb genomic regions of interest. Major candidates in these loci are Sema5a, Fmn2, Grem2, Fap, Gsg1l, Xpo6, Rabep2, Eif3c, Unc5d, and Gpr65. In particular, the refined Scc3 locus shows high concordance with the human GWAS locus that underlies hereditary mixed polyposis syndrome. These findings increase our understanding of the complex genetics of colon tumorigenesis, and provide important insights into the pathways of colorectal cancer development and might ultimately lead to more effective individually targeted cancer prevention strategies. Mol Cancer Res; 10(1); 66–74. ©2011 AACR.