Lung Tumorigenesis Research Articles

Abstract 272: Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors

Abstract Tumor suppressor Liver Kinase B1 (LKB1) activates 5’-adenosine monophosphate protein kinase (AMPK) and maintains energy homeostasis in response to energy crises. LKB1 and KRAS are the third most frequent co-mutations detected in non-small cell lung cancer (NSCLC), causing aggressive tumor growth and metastases. Unfortunately, standard treatment with RAS-RAF-MEK-ERK signaling pathway inhibitors has minimal therapeutic efficacy in LKB1-mutant KRAS-driven NSCLC. Thus, identifying a novel treatment for patients harboring co-mutations in LKB1 and KRAS is urgently needed. Autophagy degrades and recycles the building blocks for cancer cells to survive metabolic challenges. Using genetically engineered mouse models (GEMMs), we have previously demonstrated that autophagy compensates for Lkb1 loss for Kras-driven lung tumorigenesis; loss of an autophagy-essential gene Atg7 dramatically impaired tumor initiation and tumor growth in KrasG12D/+;Lkb1−/− (KL) lung tumors. This is in sharp contrast to Lkb1 wild-type (WT) (KrasG12D/+;p53−/− (KP)) tumors that are less sensitive to autophagy gene ablation. To further value our discoveries in clinical translational ability, we treated mouse lung tumor derived cell lines (TDCLs) with FDA-approved autophagy inhibitor hydroxychloroquine (HCQ) and MEK inhibitor Trametinib and found that the combination treatment displayed synergistic anti-proliferative effects in KL TDCLs compared to KP TDCLs. To elucidate the underlying mechanism of increased sensitivity of KL TDCLs to Trametinib by autophagy ablation, we performed metabolomic profiling of KL TDCLs with Trametinib, HCQ, or combination treatment and found that several glycolytic and TCA cycle intermediates, amino acids, and ATP levels were significantly upregulated upon treatment with Trametinib, which were significantly reduced by the combination treatment. In addition, the combination treatment significantly reduced mitochondrial membrane potential, basal respiration, and ATP production in KL TDCLs. In vivo studies using tumor allografts, genetically engineered mouse models (GEMMs) and patient-derived xenografts (PDXs) showed anti-tumor activity of the combination treatment on KL tumors, but not in KP tumors. Moreover, we found increased lipid peroxidation indicative of ferroptosis in KL TDCLs and KL PDX tumors with the combination treatment compared to the single agent treatments. Finally, treatment with a ferroptosis inhibitor rescued the reduced KL allograft tumor growth caused by the combination treatment. Taken together, our observations indicate that autophagy upregulation in KL tumors causes resistance to Trametinib treatment by maintaining energy homeostasis for cell survival and inhibits ferroptosis. Therefore, a combination of autophagy and MEK inhibition could be a novel therapeutic strategy to specifically treat LKB1-deficient KRAS-driven NSCLC. Citation Format: Vrushank Bhatt, Taijin Lan, Wenping Wang, Jerry Kong, Eduardo Cararo Lopes, Khoosheh Khayati, Jianming Wang, Akash Raju, Michael Rangel, Enrique Lopez, Zhixian Sherrie Hu, Xuefei Luo, Xiaoyang Su, Jyoti Malhotra, Wenwei Hu, Sharon R. Pine, Eileen White, Jessie Yanxiang Guo. Autophagy and MEK inhibition promotes ferroptosis in liver kinase B1 (Lkb1)-deficient Kras-driven lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 272.

Abstract 2856: Age-induced chronic accumulation of cortisol drives chemotherapy resistance in lung cancer

Abstract Lung cancer accounts for the largest number of cancer-associated deaths in the United States. While great strides have been made due to the introduction of immunotherapies and targeted therapies against oncogenic drivers, chemotherapies remain the standard of care for the majority of lung cancer patients. However, many patients do not respond to these treatments or relapse following an initial response. We postulate that part of the problem is the lack of consideration in preclinical research and clinical trials of the main driver of lung tumorigenesis, the aging process. Here, we show that the organismal reprogramming that occurs with aging makes lung tumors in an aged host more conducive to withstand chemotherapies. We traced this to an age-induced chronic accumulation of the stress hormone cortisol in circulation. Mechanistically our data show that chronic accumulation of cortisol drives the upregulation of metallothioneins—small, highly conserved, cysteine-rich metal-binding proteins—through activation of the glucocorticoid receptor in lung cancer cells, which in turn rewire these cells to be refractory to chemotherapies. Together, our work demonstrates a role for age-induced cortisol levels in promoting resistance to standard of care chemotherapies in lung cancer and offers a rationale for using therapeutic agents that block the glucocorticoid receptor to sensitize the average and most vulnerable lung cancer patients, the elderly, to chemotherapy. Citation Format: Devesh Raizada, Stanislav Drapela, Joanne Tejero, Nadir Sarigul, Didem Ilter, Ana Gomes. Age-induced chronic accumulation of cortisol drives chemotherapy resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2856.

Abstract 6141: Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer

Abstract Lung cancer remains the leading cause of cancer mortality. Efficacious targeted therapy options are urgently needed for lung cancer prevention and treatment. Epidermal growth factor receptor (EGFR) mutant lung cancer represents about 15% of lung cancer cases generally occurring in patients with minimal to no smoking history and with higher prevalence in females. Linkages to environmental exposures have been postulated. Our group synthesized Mito-lonidamine (Mito-LND), a mitochondrial targeted analog of lonidamine which inhibits aerobic glycolysis in cancer cells. We previously identified that Mito-LND induces mitophagy in KRAS mutant lung cells and inhibits lung tumorigenesis and brain metastasis in vivo. Yet, the cancer inhibitory capacity of Mito-LND in EGFR mutant lung cancer has not been investigated. Viability staining assessed Mito-LND induced cell death in EGFR mutant cell lines PC9 (parent) and PC9BrM3 (brain metastatic). Transcriptomic profiling was conducted following Mito-LND treatment and results analyzed in Metacore to assess molecular changes. Deconvolution analysis interrogated alterations in immune cell populations. To discern cell death mechanisms, we evaluated FDA-approved inhibitors for autophagy (chloroquine, CQ), mitophagy/apoptosis (cyclosporin A, CsA) and necrosis (Ponatinib) in EGFR mutant cell lines using viability assays with Mito-LND treatment. In PC9 cells, the IC50 for Mito-LND at 24h and 48h was 1.5µM and 0.5µM, respectively and in PC9BrM3 cells at 24h and 48h was 1.5µM and 0.7µM, respectively. These data support that EGFR mutant cell lines are >2-fold more sensitive to Mito-LND induced cell death compared to KRAS mutant lung cell lines H2030 and H2030BrM3. Transcriptomic profiling results show that Mito-LND treatment upregulated pathway maps linked to heat shock protein and HIF-1A signaling in both PC9 and PC9BrM3 cells. Process networks linked to unfolded protein response, response to hypoxia and oxidative stress, apoptosis and the immune response were upregulated with Mito-LND treatment. Deconvolution analysis revealed monocytes and macrophages were upregulated in Mito-LND treated EGFR mutant cell lines, with CD8+ T cells increased in PC9BrM3 cells and regulatory T cells increased in PC9 cells. Last, CsA significantly inhibited Mito-LND induced cell death in both cell lines, while CQ had no effect suggesting blocking apoptosis, but not late autophagy inhibits cell death induction in EGFR mutant cell lines. Further, necrosis inhibition with Ponatinib synergistically increased Mito-LND induced cell death in EGFR mutant cell lines. These results begin to define the cancer inhibitory mechanisms of Mito-LND in EGFR mutant lung cancer cells which appear to differ from those observed in KRAS mutant lung cancer cells. Future directions include dissecting apoptotic and necrosis driven Mito-LND induced cell death using additional pharmacological inhibitors and genetic approaches. Citation Format: Katherine M. Weh, Connor Howard, Yun Zhang, Jean-Jack Riethoven, Jennifer Clarke, Gang Cheng, Balaraman Kalyanaraman, Ming You, Laura Kresty. Mechanisms by which Mito-lonidamine inhibits EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6141.

The clinicopathological and prognostic significances of CDC73 expression in breast cancer: A pathological and bioinformatics analysis.

Parafibromin is a protein encoded by the oncosuppressor CDC73 gene, whose mutation results in hyperparathyroidism-jaw tumor syndrome (HPT-JT) and parathyroid carcinoma. Down-regulation of parafibromin is linked to lung, gastric, colorectal, and ovarian cancer tumorigenesis. Parafibromin expression was detected by RT-PCR, bioinformatics analysis, Western blot, and immunohistochemistry; and compared with clinicopathological characteristics of breast cancer. CDC73-related genes and pathways were analyzed using bioinformatics analysis. Parafibromin expression was increased in breast cancer compared to normal tissues at both mRNA and protein levels (p<0.05). Among triple-negative breast cancers, it was higher in basal-like 1 than basal-like 2 patients (p<0.05) and mesenchymal than immunomodulatory patients (p<0.05). CDC73 mRNA expression was positively correlated with white race, non-infiltrating immune cells, favorable luminal subtypes of PAM50, and prognosis of breast cancer patients (p<0.05). The differential genes of CDC73 were classified into enzyme inhibitors, peptidase, and keratinization by KEGG (p<0.05). Similarly, it was classified into ribosomes, TGF-β, oxidation phosphorylation, inositol phosphate metabolism, arachidonic acid metabolism, linoleic acid metabolism, ERBB, and VEGF signaling pathways by GSEA (p<0.05). The positively-correlated genes of CDC73 were involved in cell mobility, response to interferon α, nuclear pore and basket, and histone methyltransferase. The negatively-correlated genes of CDC73 were involved in the mitochondrial respiratory chain, thermogenesis, and ribosomes. Parafibromin expression was higher in invasive ductal than lobular carcinoma (p<0.05) and mucinous adenocarcinoma than others (p<0.05). Parafibromin immunoreactivity as an independent factor was positively associated with an increased overall survival rate of breast cancer patients (p<0.05). These findings suggest that up-regulation of parafibromin in breast cancer patients is closely linked to a favorable prognosis. It is involved in tumorigenesis and subsequent progression by regulating metabolism, ribosomes, and cytokines.

Targeting diacylglycerol kinase α impairs lung tumorigenesis by inhibiting cyclin D3.

Diacylglycerol kinase α (DGKA) is the first member discovered from the diacylglycerol kinase family, and it has been linked to the progression of various types of tumors. However, it is unclear whether DGKA is linked to the development of lung cancer. We investigated the levels of DGKA in the lung cancer tissues. Cell growth assay, colony formation assay and EdU assay were used to examine the effects of DGKA-targeted siRNAs/shRNAs/drugs on the proliferation of lung cancer cells in vitro. Xenograft mouse model was used to investigate the role of DGKA inhibitor ritanserin on the proliferation of lung cancer cells in vivo. The downstream target of DGKA in lung tumorigenesis was identified by RNA sequencing. DGKA is upregulated in the lung cancer cells. Functional assays and xenograft mouse model indicated that the proliferation ability of lung cancer cells was impaired after inhibiting DGKA. And cyclin D3(CCND3) is the downstream target of DGKA promoting lung cancer. Our study demonstrated that DGKA promotes lung tumorigenesis by regulating the CCND3 expression and hence it can be considered as a potential molecular biomarker to evaluate the prognosis of lung cancer patients. What's more, we also demonstrated the efficacy of ritanserin as a promising new medication for treating lung cancer.

The RNA binding protein RALY suppresses p53 activity and promotes lung tumorigenesis

The tumor suppressor p53 plays a pivotal role in tumor prevention. The activity of p53 is mainly restrained by the ubiquitin E3 ligase Mdm2. However, it is not well understood how the Mdm2-p53 pathway is intricately regulated. Here we report that the RNA binding protein RALY functions as an oncogenic factor in lung cancer. RALY simultaneously binds to Mdm2 and the deubiquitinating enzyme USP7. Via these interactions, RALY not only stabilizes Mdm2 by stimulating the deubiquitinating activity of USP7 toward Mdm2 but also increases the trans-E3 ligase activity of Mdm2 toward p53. Consequently, RALY enhances Mdm2-mediated ubiquitination and degradation of p53. Functionally, RALY promotes lung tumorigenesis, at least partially, via negative regulation of p53. These findings suggest that RALY destabilizes p53 by modulating the function of Mdm2 at multiple levels. Our study also indicates a critical role for RALY in promoting lung tumorigenesis via p53 inhibition.

The preventive role of the red gingeng ginsenoside Rg3 in the treatment of lung tumorigenesis induced by benzo(a)pyrene

Red ginseng has been used in traditional medicine for centuries in Asia. In this study, we evaluated four types of red ginseng grown in different areas (Chinese red ginseng, Korean red ginseng A, Korean red ginseng B, and Korean red ginseng C) for their ability to inhibit lung tumor formation and growth induced by the carcinogen benzo(a)pyrene (B(a)P) in A/J mice and found that Korean red ginseng B was the most effective at lowering the tumor load among the four red ginseng varieties. Moreover, we analyzed the levels of various ginsenosides (Rg1, Re, Rc, Rb2, Rb3, Rb1, Rh1, Rd, Rg3, Rh2, F1, Rk1, and Rg5) in four kinds of red ginseng extract and found that Korean red ginseng B had the highest level of ginsenoside Rg3 (G-Rg3), which suggested that G-Rg3 may play an important role in its therapeutic efficacy. This work revealed that the bioavailability of G-Rg3 was relatively poor. However, when G-Rg3 was coadministered with verapamil, a P-glycoprotein inhibitor, the G-Rg3 efflux in Caco-2 cells was lowered, the small intestinal absorption rate of G-Rg3 in the rat models was increased, the concentration levels of G-Rg3 were elevated in the intestine and plasma, and its tumor-preventive abilities in the tumorigenesis rat model induced by B(a)P were also augmented. We also found that G-Rg3 reduced B(a)P-induced cytotoxicity and DNA adduct formation in human lung cells and rescued phase II enzyme expression and activity through Nrf2 pathways, which may be the potential mechanisms underlying the inhibitory effects of G-Rg3 on lung tumorigenesis. Our study showed a potentially vital role of G-Rg3 in targeting lung tumors in murine models. The oral bioavailability of this ginsenoside was augmented by targeting P-glycoprotein, which allowed the molecule to exert its anticancer effects.

IL-36 expression is increased in NSCLC with IL-36 stimulation of lung cancer cells promoting a pro-tumorigenic phenotype

The IL-36 cytokines are a recently described subset of the IL-1 family of cytokines, and have been shown to play a role in the pathogenesis of respiratory diseases such as asthma and COPD. Given the common aetiological links between COPD and lung cancer development, as well as the involvement of other IL-1 family members in lung tumorigenesis, the aim of this work was to investigate the role of IL-36 cytokines in the pathogenesis of lung cancer. In this study we demonstrate that expression of IL-36 cytokines and receptor mRNA and protein are significantly increased in lung cancer tissue compared to adjacent non-tumour tissue. In vitro assays showed that stimulation of two lung cancer cell lines, SKMES-1 human squamous cell and LLC murine lung cancer, with IL-36R agonists resulted in increased cellular migration and proliferation. All IL-36 cytokines induced the expression of pro-inflammatory chemokines in both lung cancer cell lines with synergistic effects identified upon co-stimulation of cells with IL-17, IL-22 and TNFα. Furthermore, we report that IL-36 cytokines induce protein expression of the immune checkpoint inhibitor protein PD-L1 on lung cancer cells. Taken together, this data indicates that targeting IL-36R signalling may be a useful targeted therapy for lung cancer patients with IL-36R+ cancer cells.

Highlights from Recent Cancer Literature.

Breaking Insights| March 15 2023 Highlights from Recent Cancer Literature Author & Article Information Online Issn: 1538-7445 Print Issn: 0008-5472 ©2023 American Association for Cancer Research2023American Association for Cancer Research Cancer Res (2023) 83 (6): 805–806. https://doi.org/10.1158/0008-5472.CAN-83-6-BI Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record March 15 2023 Citation Highlights from Recent Cancer Literature. Cancer Res 15 March 2023; 83 (6): 805–806. https://doi.org/10.1158/0008-5472.CAN-83-6-BI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Although the KRAS-protein interactome has been well defined, the functional significance of these protein interactions in KRAS-mutant lung cancer tumorigenesis is poorly understood. Tang and colleagues examined the effect of candidate KRAS-interacting proteins on lung cancer growth utilizing a multiplexed somatic CRISPR/Cas9-based genome editing screen in a KRASG12D mutant mouse model. They found that wild-type HRAS and NRAS suppressed KRAS-driven lung tumorigenesis in vivo and in vitro through suppression of KRAS-KRAS binding as well as through competition for key downstream signaling pathways such as ERK. Expert Commentary: This study suggests that “RAS paralog imbalance” with decreased expression of wild-type HRAS and NRAS in the setting of mutant KRAS can promote tumorigenesis. Tang R, Shuldiner EG, Kelly M, Murray CW, Hebert JD, Andrejka L, et al. Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth. Nat Cell Biol 2023;25:159-69. Extravasation of metastatic... You do not currently have access to this content.

Clinical and genomic features of non-small cell lung cancer occurring in families.

Exposure to environmental carcinogens, such as through smoking, is a major factor in the carcinogenesis of non-small cell lung cancer (NSCLC). However, genetic factors may also contribute. To identify candidate tumor suppressor genes for NSCLC, we included 23 patients (10 related pairs and 3 individuals) with NSCLC who had other NSCLC-affected first-degree relatives in a local hospital. Exome analyses for both germline and somatic (NSCLC specimens) DNA were performed for 17 cases. Germline exome data of these 17 cases revealed that most of the short variants were identical to the variants in 14KJPN (a Japanese reference genome panel of more than 14 000 individuals) and only a nonsynonymous variant in the DHODH gene, p.A347T, was shared between a pair of NSCLC patients in the same family. This variant is a known pathogenic variant of the gene for Miller syndrome. Somatic genetic alterations in the exome data of our samples showed frequent mutations in the EGFR and TP53 genes. Principal component analysis of the patterns of 96 types of single nucleotide variants (SNVs) suggested the existence of unique mechanisms inducing somatic SNVs in each family. Delineation of mutational signatures of the somatic SNVs with deconstructSigs for the pair of germline pathogenic DHODH variant-positive cases showed that the mutational signatures of these cases included SBS3 (homologous recombination repair defect), SBS6, 15 (DNA mismatch repair), and SBS7 (ultraviolet exposure), suggesting that disordered pyrimidine production causes increased errors in DNA repair systems in these cases. Our results suggest the importance of the detailed collection of data on environmental exposure along with genetic information on NSCLC patients to identify the unique combinations that cause lung tumorigenesis in a particular family.

METTL16 promotes translation and lung tumorigenesis by sequestering cytoplasmic eIF4E2

N6-methyladenosine (m6A) plays crucial roles in regulating RNA metabolisms. METTL16 identified as a single-component methyltransferase catalyzes m6A formation in the nucleus; whether it regulates cytoplasmic RNA fate remains unknown. Here, we detected the dual localization of METTL16 in the nucleus and cytoplasm. METTL16 depletion attenuates protein synthesis, but the methyltransferase activity is not required for its translation-promoting function. Mechanistically, we identified an interactor of METTL16, eIF4E2, which represses translation by acting as a competitor of eIF4E. The METTL16-eIF4E2 interaction impedes the recruitment of eIF4E2 to 5' cap structure, promoting the cap recognition by eIF4E and selective protein synthesis. Depletion of METTL16 suppresses lung tumorigenesis by downregulating the translation of key oncogenes. Collectively, our study reports a role of METTL16 in modulating translation and provides a therapeutic target for lung cancer treatment.

SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis.

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers.

Discovery of potential biomarkers for lung cancer classification based on human proteome microarrays using Stochastic Gradient Boosting approach.

Early identification of lung cancer (LC) will considerably facilitate the intervention and prevention of LC. The human proteome micro-arrays approach can be used as a "liquid biopsy" to diagnose LC to complement conventional diagnosis, which needs advanced bioinformatics methods such as feature selection (FS) and refined machine learning models. A two-stage FS methodology by infusing Pearson's Correlation (PC) with a univariate filter (SBF) or recursive feature elimination (RFE) was used to reduce the redundancy of the original dataset. The Stochastic Gradient Boosting (SGB), Random Forest (RF), and Support Vector Machine (SVM) techniques were applied to build ensemble classifiers based on four subsets. The synthetic minority oversampling technique (SMOTE) was used in the preprocessing of imbalanced data. FS approach with SBF and RFE extracted 25 and 55 features, respectively, with 14 overlapped ones. All three ensemble models demonstrate superior accuracy (ranging from 0.867 to 0.967) and sensitivity (0.917to 1.00) in the test datasets with SGB of SBF subset outperforming others. The SMOTE technique has improved the model performance in the training process. Three of the top selected candidate biomarkers (LGR4, CDC34, and GHRHR) were highly suggested to play a role in lung tumorigenesis. A novel hybrid FS method with classical ensemble machine learning algorithms was first used in the classification of protein microarray data. The parsimony model constructed by the SGB algorithm with the appropriate FS and SMOTE approach performs well in the classification task with higher sensitivity and specificity. Standardization and innovation of bioinformatics approach for protein microarray analysis need further exploration and validation.

Precision immunointerception of EGFR-driven tumorigenesis for lung cancer prevention.

Epidermal growth factor receptor (EGFR) mutations occur in about 50% of lung adenocarcinomas in Asia and about 15% in the US. EGFR mutation-specific inhibitors have been developed and made significant contributions to controlling EGFR mutated non-small cell lung cancer. However, resistance frequently develops within 1 to 2 years due to acquired mutations. No effective approaches that target mutant EGFR have been developed to treat relapse following tyrosine kinase inhibitor (TKI) treatment. Vaccination against mutant EGFR is one area of active exploration. In this study, we identified immunogenic epitopes for the common EGFR mutations in humans and formulated a multi-peptide vaccine (Emut Vax) targeting the EGFR L858R, T790M, and Del19 mutations. The efficacy of the Emut Vax was evaluated in both syngeneic and genetic engineered EGFR mutation-driven murine lung tumor models with prophylactic settings, where the vaccinations were given before the onset of the tumor induction. The multi-peptide Emut Vax effectively prevented the onset of EGFR mutation-driven lung tumorigenesis in both syngeneic and genetically engineered mouse models (GEMMs). Flow cytometry and single-cell RNA sequencing were conducted to investigate the impact of Emut Vax on immune modulation. Emut Vax significantly enhanced Th1 responses in the tumor microenvironment and decreased suppressive Tregs to enhance anti-tumor efficacy. Our results show that multi-peptide Emut Vax is effective in preventing common EGFR mutation-driven lung tumorigenesis, and the vaccine elicits broad immune responses that are not limited to anti-tumor Th1 response.

The UBE2C/CDH1/DEPTOR axis is an oncogene and tumor suppressor cascade in lung cancer cells.

Ubiquitin-conjugating enzyme E2C (UBE2C) mediates ubiquitylation chain formation via the K11 linkage. While previous in vitro studies showed that UBE2C plays a growth-promoting role in cancer cell lines, the underlying mechanism remains elusive. Still unknown is whether and how UBE2C plays a promoting role in vivo. Here we report that UBE2C was indeed essential for growth and survival of lung cancer cells harboring Kras mutations, and UBE2C was required for KrasG12D-induced lung tumorigenesis, since Ube2c deletion significantly inhibited tumor formation and extended the lifespan of mice. Mechanistically, KrasG12D induced expression of UBE2C, which coupled with APC/CCDH1 E3 ligase to promote ubiquitylation and degradation of DEPTOR, leading to activation of mTORC signaling. Importantly, DEPTOR levels fluctuated during cell cycle progression in a manner dependent on UBE2C and CDH1, indicating their physiological connection. Finally, Deptor deletion fully rescued the tumor inhibitory effect of Ube2c deletion in the KrasG12D lung tumor model, indicating a causal role of Deptor. Taken together, our study shows that the UBE2C/CDH1/DEPTOR axis forms an oncogene and tumor suppressor cascade that regulates cell cycle progression and autophagy and validates UBE2C an attractive target for lung cancer associated with Kras mutations.

Critical Role of the Sulfiredoxin-Peroxiredoxin IV Axis in Urethane-Induced Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC), the most common type of lung cancer, etiologically associates with tobacco smoking which mechanistically contributes to oxidative stress to facilitate the occurrence of mutations, oncogenic transformation and aberrantly activated signaling pathways. Our previous reports suggested an essential role of Sulfiredoxin (Srx) in promoting the development of lung cancer in humans, and was causally related to Peroxiredoxin IV (Prx4), the major downstream substrate and mediator of Srx-enhanced signaling. To further explore the role of the Srx-Prx4 axis in de novo lung tumorigenesis, we established Prx4-/- and Srx-/-/Prx4-/- mice in pure FVB/N background. Together with wild-type litter mates, these mice were exposed to carcinogenic urethane and the development of lung tumorigenesis was evaluated. We found that disruption of the Srx-Prx4 axis, either through knockout of Srx/Prx4 alone or together, led to a reduced number and size of lung tumors in mice. Immunohistological studies found that loss of Srx/Prx4 led to reduced rate of cell proliferation and less intratumoral macrophage infiltration. Mechanistically, we found that exposure to urethane increased the levels of reactive oxygen species, activated the expression of and Prx4 in normal lung epithelial cells, while knockout of Prx4 inhibited urethane-induced cell transformation. Moreover, bioinformatics analysis found that the Srx-Prx4 axis is activated in many human cancers, and their increased expression is tightly correlated with poor prognosis in NSCLC patients.

Targeting polymerase θ impairs tumorigenesis and enhances radiosensitivity in lung adenocarcinoma.

Radioresistance remains a major obstacle to efficacious radiotherapy in non-small-cell lung cancer (NSCLC). DNA replication proteins are novel targets for radiosensitizers. POLQ is a DNA polymerase involved in DNA damage response and repair. We found that POLQ is overexpressed in NSCLC and is clinically correlated with high tumor stage, poor prognosis, increased tumor mutational burden, and ALK and TP5 mutation status; POLQ inhibition impaired lung tumorigenesis. Notably, POLQ expression was higher in radioresistant lung cancer cells than in wild-type cancer cells. Moreover, POLQ expression was further increased in radioresistant cells after radiation. Enhanced radioresistance is through a prolonged G2/M phase and faster repair of DNA damage, leading to reduced radiation-induced apoptosis. Novobiocin (NVB), a POLQ inhibitor, specifically targeted cancer cells. Genetic knockdown of POLQ or pharmacological inhibition by NVB decreased radioresistance in lung adenocarcinoma while causing little toxicity to normal pulmonary epithelial cells. In conclusion, POLQ is a promising and practical cancer-specific target to impair tumorigenesis and enhance radiosensitivity in NSCLC.

Effect of a Cyclooxygenase-2 Inhibitor in Combination with (-)-Epigallocatechin Gallate or Polyphenon E on Cisplatin-Induced Lung Tumorigenesis in A/J Mice.

We investigated the effects of celecoxib combined with (-)-epigallocatechin-3-gallate (EGCG) or polyphenon E in a cisplatin-induced lung tumorigenesis model. Four-week-old female A/J mice were divided into seven groups: (i) Control, (ii) 150 mg/kg celecoxib (150Cel), (iii) 1,500 mg/kg celecoxib (1500Cel), (iv) EGCG+150 mg/kg celecoxib (EGCG+150Cel), (v) EGCG+1,500 mg/kg celecoxib (EGCG+1500Cel), (vi) polyphenon E+150 mg/kg celecoxib (PolyE+150Cel), and (vii) polyphenon E+1,500 mg/kg celecoxib (PolyE+1500Cel). All mice were administered cisplatin (1.62 mg/kg of body weight, i.p.) 1×/week for 10 weeks and sacrificed at week 30; the numbers of tumors on the lung surface were then determined. The tumor incidence and multiplicity (no. of tumors/mouse, mean±SD) were respectively 95% and 2.15±1.50 in Control, 95% and 2.10±1.29 in 150Cel, 86% and 1.67±1.20 in 1500Cel, 71% and 1.38±1.24 in EGCG+150Cel, 67% and 1.29±1.38 in EGCG+1500Cel, 80% and 1.95±1.36 in PolyE+150Cel, and 65% and 1.05±0.10 in PolyE+1500Cel. The combination of high-dose celecoxib with EGCG or polyphenon E significantly reduced multiplicity in cisplatin-induced lung tumors.

Long-term exposure to house dust mites accelerates lung cancer development in mice

BackgroundIndividuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI). MethodsThe effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C–C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM. ResultsChronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment.ConclusionsCollectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

Abstract IA022: Age-induced metabolic reprogramming: A bridge between the aging process and tumorigenesis

Abstract Lung cancer accounts for the largest number of cancer-associated deaths in the United States. While great strides have been made due to the introduction of immunotherapies and targeted therapies against oncogenic drivers, chemotherapies remain the standard of care for the majority of lung cancer patients. However, many patients do not respond to these treatments or relapse following an initial response. We postulate that part of the problem is the lack of consideration in preclinical research and clinical trials of the main driver of lung tumorigenesis, the aging process. Here, we show that the organismal metabolic reprogramming that occurs with aging endows cancer cells with pro-aggressive properties favoring progression into metastatic disease through, at least in part, the accumulation in the circulation of methylmalonic acid, a by-product of vitamin B12 deficiency. On the other hand, we also show that this age-induced organismal metabolic reprogramming makes lung tumors intrinsically more resistant to chemotherapies through the chronic upregulation of glucocorticoid hormones. Together, our work demonstrates a role for age-induced systemic metabolic reprogramming in shaping the tumorigenic process toward more aggressive, treatment-resistant disease and offers a rationale for using interventions that ameliorate the metabolic deregulation that occurs with age in the treatment of age-related cancers. Citation Format: Ana P. Gomes. Age-induced metabolic reprogramming: A bridge between the aging process and tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA022.