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One of the most fundamental and challenging questions in the cancer field is how immunity in patients with cancer is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identify the transcription factor STAT3 as the culprit responsible for this pathogenic event in lung cancer development. We found that antitumor type 1 CD4+ T-helper (Th1) cells and CD8+ T cells were directly counter balanced in lung cancer development with tumor-promoting myeloid-derived suppressor cells (MDSCs) and suppressive macrophages, and that activation of STAT3 in MDSCs and macrophages promoted tumorigenesis through pulmonary recruitment and increased resistance of suppressive cells to CD8+ T cells, enhancement of cytotoxicity toward CD4+ and CD8+ T cells, induction of regulatory T cell (Treg), inhibition of dendritic cells (DC), and polarization of macrophages toward the M2 phenotype. The deletion of myeloid STAT3 boosted antitumor immunity and suppressed lung tumorigenesis. These findings increase our understanding of immune programming in lung tumorigenesis and provide a mechanistic basis for developing STAT3-based immunotherapy against this and other solid tumors. Cancer Immunol Res; 5(3); 257–68. ©2017 AACR.

Supplementary Figure S1. Decreased survival rate of lung cancer patients who are older than 65 and with high STAT3 activation in TAMs Supplementary Figure S2. STAT3 knockout in myeloid cells in STAT3DMye mice Supplementary Figure S3. STAT3 knockout in myeloid cells in STAT3DMye mice Supplementary Figure S4. Decreased lung tumorigenesis in urethane-treated STAT3DMye mice Supplementary Figure S5. Same pathology of the lung tumors in WT and STAT3DMye mice treated with urethane Supplementary Figure S6. Different changes in the total numbers of lung macrophages, neutrophils and lymphocytes in urethane-treated STAT3DMyc mice Supplementary Figure S7. Increased M1 macrophages but decreased M2 macrophages in the lungs of urethane-treated STAT3DMye mice Supplementary Figure S8. Lung myeloid and lymphoid cell changes in STAT3DMye mice treated with urethane Supplementary Figure S9. Expression changes of chemokines in mouse lungs by urethane Supplementary Figure S10. T-cell apoptosis by MDSCs Supplementary Figure S11. Defect in T-cell apoptosis induction by STAT3 KO macrophages Supplementary Figure S12. No obvious effect on T-cell proliferation and lung recruitment by myeloid STAT3 deletion Supplementary Figure S13. Treg cell differentiation by WT macrophages but not by STAT3 KO macrophages Supplementary Figure S14. Reversal of the decreased lung tumorigenesis in urethane-treated STAT3DMye mice by CD8 deletion Supplementary Figure S15. Quantification of the iNOS and arginase staining in the Figure 7F Supplementary Figure S16. Reduced STAT3 activation in lung tumor cells in STAT3DMye mice Supplementary Table S1. Antibodies used in this study Supplementary Table S2. Clinicopathological characteristics of lung cancer patients Supplementary Table S3. Primer pairs used for real-time PCR in the study

Background In patients with non-small cell lung cancer (NSCLC), 10%-40% will eventually develop brain metastases. We present the clinicopathologic, genomic, and biomarker landscape of a large cohort of NSCLC brain metastases (NSCLC-BM) samples. Materials and Methods We retrospectively analyzed 3035 NSCLC-BM tested with comprehensive genomic profiling (CGP) during routine clinical care. In addition, we compared the NSCLC-BM to a separate cohort of 7277 primary NSCLC (pNSCLC) specimens. Finally, we present data on 67 paired patients with NSCLC-BM and pNSCLC. Results Comprehensive genomic profiling analysis of the 3035 NSCLC-BMs found that the most frequent genomic alterations (GAs) were in the TP53, KRAS, CDKN2A, STK11, CDKN2B, EGFR, NKX2-1, RB1, MYC, and KEAP1 genes. In the NSCLC-BM cohort, there were significantly higher rates of several targetable GAs compared with pNSCLC, including ALK fusions, KRAS G12C mutations, and MET amplifications; and decreased frequency of MET exon14 skipping mutations (all P < .05). In the subset of NSCLC-BM (n = 1063) where concurrent PD-L1 immunohistochemistry (IHC) was performed, 54.7% of the patients with NSCLC-BM were eligible for pembrolizumab based on PD-L1 IHC (TPS ≥ 1), and 56.9% were eligible for pembrolizumab based on TMB-High status. In addition, in a series 67 paired pNSCLC and NSCLC-BM samples, 85.1% (57/67) had at least one additional GA discovered in the NSCLC-BM sample when compared with the pNSCLC sample. Conclusions Herein, we defined the clinicopathologic, genomic, and biomarker landscape of a large cohort of patients with NSCLC-BM which can help inform study design of future clinical studies for patients with NSCLC with BM. In certain clinical situations, metastatic NSCLC brain tissue or cerebral spinal fluid specimens may be needed to fully optimize personalized treatment.

Purpose: Steroid hormones and growth factors affect lung cancer, and it is possible they act in concert to influence patient outcome.Experimental Design: Primary lung tumors and normal lung tissue were analyzed for expression and localization of estrogen receptor α and β-1 (ERα and ERβ), aromatase, progesterone receptor (PR), and epidermal growth factor receptor (EGFR).Results: Tumors expressed higher levels of ERβ compared to matched normal lung, whereas the reverse was true of PR. High cytoplasmic ERβ expression was identified as an independent negative prognostic predictor of overall survival (OS; HR = 1.67), and low total PR was identified as an independent negative predictor of time to progression (TTP; HR = 1.59). After adjusting for stage, age, sex, and smoking, combined high cytoplasmic ERβ and low total PR showed enhanced effects on OS (HR = 2.64) and on TTP (HR = 6.02). Further effects on OS were observed when EGFR expression was included (HR = 5.32). Patients with low cytoplasmic ERβ, low aromatase, low EGFR, and high total PR had shorter OS than patients with the opposite pattern (HR = 6.60). Contribution of these markers to survival showed no significant sex differences in a multivariable model. ERα was elevated in tumors but was not predictive of survival, and appears to represent a variant ERα protein that is only recognized by a C-terminal antibody.Conclusions: Hormonal and EGFR pathways together may contribute to lung cancer prognosis. Lung tumors with high ERβ-1/low PR may define patients with aggressive biology. A validation study is necessary to fully assess the predictive value of these markers. Clin Cancer Res; 17(1); 154–64. ©2010 AACR.

Supplementary Data from HGF and c-Met Participate in Paracrine Tumorigenic Pathways in Head and Neck Squamous Cell Cancer

PDF file - 473K, Phospho-Met immunohistochemistry from representative VC-2 and DA-Src-5 tumor xenografts. Quantitative data of 5 samples from each treatment group. P

PDF file - 36K, After 48 hrs of incubation, the conditioned media was harvested from normal lung fibroblasts (NLFB, positive control), 201T lung tumor cells (negative control), VC-2, and DA-Src-5 cell lines. Each conditioned medium sample was concentrated from 4ml to 500μl. ELISA was performed with the Quantikine� Immunoassay Kit of human HGF. HGF concentration in the media was normalized to its respective protein concentration from total cell lysates. Each reaction was performed in triplicate.

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Purpose: We determined hepatocyte growth factor (HGF) and c-Met expression and signaling in human head and neck squamous cell carcinoma (HNSCC) cells and primary tissues and tested the ability of c-Met tyrosine kinase inhibitors (TKI) to block HGF-induced biological signaling.Experimental Design: Expression and signaling were determined using immunoblotting, ELISA, and immunohistochemistry. Biological end points included wound healing, cell proliferation, and invasion. c-Met TKIs were tested for their ability to block HGF-induced signaling and biological effects in vitro and in xenografts established in nude mice.Results: c-Met was expressed and functional in HNSCC cells. HGF was secreted by HNSCC tumor-derived fibroblasts, but not by HNSCC cells. Activation of c-Met promoted phosphorylation of AKT and mitogen-activated protein kinase as well as release of the inflammatory cytokine interleukin-8. Cell growth and wound healing were also stimulated by HGF. c-Met TKIs blocked HGF-induced signaling, interleukin-8 release, and wound healing. Enhanced invasion of HNSCC cells induced by the presence of tumor-derived fibroblasts was completely blocked with a HGF-neutralizing antibody. PF-2341066, a c-Met TKI, caused a 50% inhibition of HNSCC tumor growth in vivo with decreased proliferation and increased apoptosis within the tumors. In HNSCC tumor tissues, both HGF and c-Met protein were increased compared with expression in normal mucosa.Conclusions: These results show that HGF acts mainly as a paracrine factor in HNSCC cells, the HGF/c-Met pathway is frequently up-regulated and functional in HNSCC, and a clinically relevant c-Met TKI shows antitumor activity in vivo. Blocking the HGF/c-Met pathway may be clinically useful for the treatment of HNSCC.

PDF file - 31K, DA-Src cells and VC cells had similar sensitivity towards cisplatin treatment. Cells were treated with cisplatin (0.1μM, 1.0μM and 5.0μM) for 72 hrs, followed by MTT assay. Cumulative data from 3 independent experiments are shown. No statistical significant difference was observed at any dose between the VC and DA-Src groups (P>0.1, ns.).

The hepatocyte growth factor (HGF)/c-Met signaling pathway is involved in lung tumor growth and progression, and agents that target this pathway have clinical potential for lung cancer treatment. L2G7, a single potent anti-human HGF neutralizing monoclonal antibody, showed profound inhibition of human HGF-induced phosphorylated mitogen-activated protein kinase induction, wound healing, and invasion in lung tumor cells in vitro. Transgenic mice that overexpress human HGF in the airways were used to study the therapeutic efficacy of L2G7 for lung cancer prevention. Mice were treated with the tobacco carcinogen, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, over 4 weeks. Beginning at week 3, i.p. treatment with 100 μg L2G7 or isotype-matched antibody control, 5G8, was initiated and continued through week 15. The mean number of tumors per mouse in the L2G7-treated group was significantly lower than in the control group (1.58 versus 3.19; P = 0.0005). Proliferative index was decreased by 48% (P = 0.013) in tumors from L2G7-treated mice versus 5G8-treated mice, whereas extent of apoptosis was increased in these same tumors by 5-fold (P = 0.0013). Phosphorylated mitogen-activated protein kinase expression was also significantly decreased by 84% in tumors from L2G7-treated mice versus 5G8-treated mice (P = 0.0003). Tumors that arose in HGF transgenic animals despite L2G7 treatment were more likely to contain mutant K-ras, suggesting that targeting the HGF/c-Met pathway may not be as effective if downstream signaling is activated by a K-ras mutation. These preclinical results show that blocking the HGF/c-Met interaction with a single monoclonal antibody delivered systemically can have profound inhibitory effects on development of lung tumors. [Mol Cancer Ther 2008;7(7):1913–22]

Supplementary Fig. S1 from Therapeutic targeting of human hepatocyte growth factor with a single neutralizing monoclonal antibody reduces lung tumorigenesis

Supplementary Information includes supplemental experimental procedures, description of gene regulatory network, two tables, eight figures and a supplementary video. Supplementary Table S1. List of primers used Supplementary Table S2. Parameters used in the ODEs Supplementary Figure S1: Large T47D microtumors exhibit collective migration, hypoxia and aggressive phenotype. Supplementary video showing migration of a microtumor Supplementary Figure S2: Treatment of 150 and 600μm microtumors with conditioned media of 150μm microtumors (150/CM). Supplementary Figure S3: Morphology and multi-parameter flow cytometry on breast cancer metastatic pleural effusion cells. Supplementary Figure S4: Large microtumors of patient-derived primary cancer cells and xenograft cells showed migratory behavior. Supplementary Figure S5: Integrated regulatory network of tumor secretome factors and signaling molecules involved in microtumor migration. Supplementary Figure S6: HIF-1α inhibition at later time points does not inhibit migration of 600μm microtumors. Supplementary Figure S7: Effect of MMP inhibitor treatment on the collective migration of large microtumors of T47D cell line and patient-derived metastatic breast cancer (mBC) cells. Supplementary Figure S8: Effect of Anti-sE-CAD antibody treatment on the collective migration of large microtumors of T47D cells and patient-derived metastatic breast cancer (mBC) cells.

Targeting microenvironmental factors that foster migratory cell phenotypes is a promising strategy for halting tumor migration. However, lack of mechanistic understanding of the emergence of migratory phenotypes impedes pharmaceutical drug development. Using our three-dimensional microtumor model with tight control over tumor size, we recapitulated the tumor size–induced hypoxic microenvironment and emergence of migratory phenotypes in microtumors from epithelial breast cells and patient-derived primary metastatic breast cancer cells, mesothelioma cells, and lung cancer xenograft cells. The microtumor models from various patient-derived tumor cells and patient-derived xenograft cells revealed upregulation of tumor-secreted factors, including matrix metalloproteinase-9 (MMP9), fibronectin (FN), and soluble E-cadherin, consistent with clinically reported elevated levels of FN and MMP9 in patient breast tumors compared with healthy mammary glands. Secreted factors in the conditioned media of large microtumors induced a migratory phenotype in nonhypoxic, nonmigratory small microtumors. Subsequent mathematical analyses identified a two-stage microtumor progression and migration mechanism whereby hypoxia induces a migratory phenotype in the initialization stage, which then becomes self-sustained through a positive feedback loop established among the tumor-secreted factors. Computational and experimental studies showed that inhibition of tumor-secreted factors effectively halts microtumor migration despite tumor-to-tumor variation in migration kinetics, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. In summary, we show that targeting temporal dynamics of evolving microenvironments, especially tumor-secreted factors during tumor progression, can halt tumor migration.Significance:This study uses state-of-the-art three-dimensional microtumor models and computational approaches to highlight the temporal dynamics of tumor-secreted microenvironmental factors in inducing tumor migration.

Purpose: Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.Experimental Design: Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways.Results: Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo.Conclusions: Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC. Clin Cancer Res; 17(13); 4425–38. ©2011 AACR.

Purpose: Strategies to inhibit the EGF receptor (EGFR) using the tyrosine kinase inhibitor erlotinib have been associated with limited clinical efficacy in head and neck squamous cell carcinoma (HNSCC). Co-activation of alternative kinases may contribute to erlotinib resistance.Experimental Design: We generated HNSCC cells expressing dominant-active c-Src (DA-Src) to determine the contribution of c-Src activation to erlotinib response.Results: Expression of DA-Src conferred resistance to erlotinib in vitro and in vivo compared with vector-transfected control cells. Phospho-Met was strongly upregulated by DA-Src, and DA-Src cells did not produce hepatocyte growth factor (HGF). Knockdown of c-Met enhanced sensitivity to erlotinib in DA-Src cells in vitro, as did combining a c-Met or c-Src inhibitor with erlotinib. Inhibiting EGFR resulted in minimal reduction of phospho-Met in DA-Src cells, whereas complete phospho-Met inhibition was achieved by inhibiting c-Src. A c-Met inhibitor significantly sensitized DA-Src tumors to erlotinib in vivo, resulting in reduced Ki67 labeling and increased apoptosis. In parental cells, knockdown of endogenous c-Src enhanced sensitivity to erlotinib, whereas treatment with HGF to directly induce phospho-Met resulted in erlotinib resistance. The level of endogenous phospho-c-Src in HNSCC cell lines was also significantly correlated with erlotinib resistance.Conclusions: Ligand-independent activation of c-Met contributes specifically to erlotinib resistance, not cetuximab resistance, in HNSCC with activated c-Src, where c-Met activation is more dependent on c-Src than on EGFR, providing an alternate survival pathway. Addition of a c-Met or c-Src inhibitor to erlotinib may increase efficacy of EGFR inhibition in patients with activated c-Src. Clin Cancer Res; 19(2); 380–92. ©2012 AACR.

PDF file - 38K, HNSCC cell line

Early detection of lung cancer is essential to reduce lung cancer-related morbidity and mortality. Despite significant efforts, current approaches suffer from high false-positive rates or limited sensitivity. There is a critical need for non-invasive, cost-effective technologies that detect the early changes in lung carcinogenesis with high sensitivity and specificity. Abnormal chromatin structure is one of the hallmarks of cancer cells and is independent of molecular pathways making it an attractive target for early cancer detection. In this study, we assessed the use of super-resolution stochastic optical reconstruction microscopy (STORM) to detect abnormal heterochromatin disruption in lung carcinogenesis. First, utilizing a mouse model of tobacco carcinogen (NNK)-induced lung adenocarcinoma, we analyzed heterochromatin structure in the lungs of NNK-treated mice at 8, 15 and 20 weeks post-NNK exposure together with non-treated control mice at the same time points. Super-resolution images of NNK-induced lung lesions at all time points showed significant disruption of condensed heterochromatin nanodomains compared to normal alveolar epithelial cells from the control mice, consistent with our data from other tumor types. Importantly, the normal-appearing bronchial epithelial cell nuclei from NNK-treated mice also showed significant heterochromatin disruption in NNK-treated mice at 15 and 20 weeks (P< 0.0001), demonstrating that this method can detect early changes. Next, we sought to confirm this in humans. Super-resolution imaging showed that, compared to normal alveolar and bronchial epithelial cells from control subjects without cancer, the heterochromatin structure was more disrupted in tumor cells from patients with lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). We also observed significant disrupted heterochromatin structure in tumor-adjacent normal tissue at the tumor margin. Lastly, we performed a pilot study on bronchial epithelial cells isolated from sputum samples from 11 cigarette smokers with or without lung cancer. The heterochromatin structure in the bronchial epithelial cells collected from lung cancer patients at the time of diagnosis was significantly more disrupted than those from smokers without lung cancer (P= 0.004). Our results support disrupted heterochromatin structure as an early event in lung carcinogenesis and demonstrate the feasibility of utilizing super-resolution imaging of heterochromatin structure for early detection of lung cancer using sputum samples. Citation Format: Laura P. Stabile, Xuejiao Sun, Jianquan Xu, Beatriz Kanterewicz, David O. Wilson, Sanja Dacic, Lora H. Rigatti, Brenda Diergaarde, Yang Liu. Super-resolution imaging of heterochromatin structure detects early events 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 777.

Non-small cell lung cancer (NSCLC) has the highest incidence of brain metastases (BM) with almost 40% of lung cancer patients developing BM throughout the course of their disease. The presence of BM portends an extremely poor prognosis even when extracranial disease is controlled and remains a major clinical problem. To date, there are no BM-specific targeted therapies available. MET is a receptor tyrosine kinase that, upon binding hepatocyte growth factor (HGF), mediates proliferation, epithelial-mesenchymal transition, invasion, angiogenesis and metastasis. The MET pathway has emerged as a targetable oncogenic driver of NSCLC BM; however, almost half of patients with MET alterations fail to respond to MET tyrosine kinase inhibitors (TKIs). Interestingly, we identified a significant enrichment of MET amplification in lung adenocarcinoma (LUAD) BM (16%) compared to primary LUAD (3%) or liver metastases (5%). Subsequent RNA-sequencing and Gene Set Enrichment Analysis of MET-amplified and non-MET amplified LUAD BM identified many dysregulated pathways including those involved in cellular metabolism. In contrast to previous reports in melanoma which found that oxidative phosphorylation was the dominant metabolic pathway in BM, we found that genes involved in glycolysis and glutamine catabolism were increased in the high MET expressing cell lines compared to low MET expressing cell lines. We confirmed glycolytic pathway up-regulation by evaluating the activity of hexokinase 1 and 2 (HK1, HK2), glutaminase (GLS), and lactate dehydrogenase (LDHA) in a MET-amplified metastatic line (H1993) compared to a MET wild-type NSCLC cell line (H2073) derived from the same patient. Further, bioenergetic analysis revealed that H1993 cells were more metabolically active, generated higher levels of ATP, and exhibited increased glycolysis compared to H2073 cells. The enhanced metabolic activity of H1993 cells increased their susceptibility to glucose deprivation and metabolic inhibitors, supporting that metabolic reprogramming is important for MET-driven disease. Additionally, untargeted metabolomics of MET amplified compared to non-MET amplified cells identified alterations in numerous amino acid catabolism pathways, including glutathione biosynthesis among other novel pathways. Treatment with the MET TKI capmatinib similarly reduced glycolysis-associated gene expression, oxygen consumption, extracellular acidification, and amino acid catabolism generating a metabolic phenotype more comparable to non-MET amplified NSCLC. Together, our data show MET-amplified NSCLC BM undergo metabolic reprogramming, which can be inhibited to suppress tumor growth. Current and future experiments are exploring whether we can specifically target MET altered BM with glycolytic inhibitors. Citation Format: Kasey R. Cargill, Sanja Dacic, Riyue Bao, Bharathri Sivakama, Eric S. Goetzman, Steven J. Mullett, Stacy G. Wendell, Sameer Agnihotri, Laura P. Stabile, Timothy F. Burns. Targeting metabolic vulnerabilities in MET-driven lung cancer brain metastases. [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 6042.

K-ras mutant lung adenocarcinoma (LUAD) is the most common type of lung cancer, displays abysmal prognosis and is tightly linked to tumor-promoting inflammation, which is increasingly recognized as a target for therapeutic intervention. We have recently shown a gender-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant LUAD. The absence of epithelial Stat3 in male K-ras mutant mice (LR/Stat3Δ/Δ mice) promoted tumorigenesis and induced a nuclear factor-kappaB (NF-κB)-driven pro-tumor immune response while reducing tumorigenesis and enhancing anti-tumor immunity in female counterparts. In the present study, we manipulated estrogen and NF-κB signaling to study the mechanisms underlying this intriguing gender-disparity. In LR/Stat3Δ/Δ females, estrogen deprivation by bilateral oophorectomy resulted in higher tumor burden, an induction of NF-κB-driven immunosuppressive response, and reduced anti-tumor cytotoxicity, whereas estrogen replacement reversed these changes. On the other hand, exogenous estrogen in males successfully inhibited tumorigenesis, attenuated NF-κB-driven immunosuppression and boosted anti-tumor immunity. Mechanistically, genetic targeting of epithelial NF-κB activity resulted in reduced tumorigenesis and enhanced the anti-tumor immune response in LR/Stat3Δ/Δ males, but not females. Our data suggest that estrogen exerts a context-specific anti-tumor effect through inhibiting NF-κB-driven tumor-promoting inflammation and provide insights into developing novel personalized therapeutic strategies for K-ras mutant LUAD.

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes. Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells. Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

High ERβ/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.

Former smokers have an elevated risk of lung cancer and account for a large proportion of newly diagnosed lung cancer. Former smokers with airway dysplasia have received modest benefit in randomized chemoprevention trials, while active smokers have not. More effective chemoprevention agents for former smokers at risk for lung cancer are needed. We previously reported that a cyclic double-stranded DNA oligonucleotide STAT3 decoy (CS3D) administered intravenously (IV) had strong anti-tumor effects in lung cancer xenograft models and was also effective as a chemoprevention agent using a tobacco carcinogen (NNK)-induced lung cancer mouse model that mimics “ex-smokers”. CS3D prevents the binding of activated STAT3 dimers to the promoter of target genes and induces p-STAT3 degradation. Our goal was to develop a clinically applicable direct delivery method of CS3D to the lungs. Using fluorescently tagged CS3D, we evaluated the feasibility and determined the optimal dosing schedule of CS3D administered intratracheally (IT, a mimic of human inhalation) compared to IV in mice. IT delivery 3 times/week provided a high initial drug level that was cleared from the lungs within a few hours, while IV dosing provided lower initial drug levels but persisted up to 8 hrs, with CS3D accumulation over time in the lungs. While the pharmacodynamic effect was also greater with systemic dosing, IT delivery produced inhibition of the STAT3 pathway. Phosphorylated STAT3, VEGF, IL6 and Ki67 expression in NNK-induced lung preneoplasias was down-modulated by IT CS3D delivery after 4 weeks of treatment compared to the mutant oligonucleotide (CS3M), confirming our previous findings with IV delivery. We also demonstrated that CS3D delivered IT altered the pulmonary environment by promoting a pro-inflammatory, anti-tumor response in myeloid cells of the lung directly, or by acting on tumor and stromal components to establish an immune-reactive tumor-microenvironment. No toxicities were found during IT or IV treatment and no organ abnormalities were detected by either regimen. A long-term chemoprevention study of CS3D delivered IT in the ex-smoker lung cancer murine model is underway. Our findings suggest that blocking STAT3 may be a useful strategy for lung cancer prevention, and may involve both inhibition of oncogenic signaling and enhanced anti-tumor immunity. Citation Format: Jill M. Siegfried, Adam C. Soloff, Autumn Gaither Davis, Amy A. Powers, Seth H. Eisenberg, Laura P. Stabile. Targeting STAT3 for lung cancer prevention [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 A019.

Estrogen receptors (ERs) are known to play an important role in the proper development of estrogen-sensitive organs, as well as in the development and progression of various types of cancer. ERα, the first ER to be discovered, has been the focus of most cancer research, especially in the context of breast cancer. However, ERβ expression also plays a significant role in cancer pathophysiology, notably its seemingly protective nature and loss of expression with oncogenesis and progression. Although ERβ exhibits antitumor activity in breast, ovarian, and prostate cancer, its expression is associated with disease progression and worse prognosis in lung cancer. The function of ERβ is complicated by the presence of multiple isoforms and single nucleotide polymorphisms, in addition to tissue-specific functions. This mini-review explores current literature on ERβ and its mechanism of action and clinical implications in breast, ovarian, prostate, and lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide, with a 5-year survival rate of about 18%. Thus, there is a great need for novel therapeutic approaches to treat non-small-cell lung cancer (NSCLC). Immune checkpoint inhibitors (ICIs) have improved outcomes for a subset of patients, especially those with high programmed death-ligand 1 expression and/or high tumor mutational burden, but have failed in the majority of patients. Increasing evidence suggests that the estrogen signaling pathway may be a therapeutic target in metastatic NSCLC and that the estrogen pathway may play a role in sex-based responses to ICIs. This report will review the epidemiologic, preclinical and clinical data on the estrogen pathway in NSCLC, its implications in sex-based responses to ICIs and the potential use of antiestrogen therapy in combination with ICIs.

Background The epidermal growth factor receptor (EGFR) monoclonal IgG1 antibody cetuximab is approved for first-line treatment of recurrent and metastatic (R/M) HNSCC as a part of the standard of care EXTREME regimen (platinum/5-fluorouracil/cetuximab). This regimen has relatively high response and disease control rates but is generally not curative and many patients will experience recurrent disease and/or metastasis. Therefore, there is a great need to identify predictive biomarkers for recurrence and disease progression in cetuximab-treated HNSCC patients to facilitate patient management and allow for treatment modification. The goal of this work is to assess the potential of activating interleukin-1 (IL-1) ligands (IL-1 alpha [IL-1α], IL-1 beta [IL-1β]) as predictive biomarkers of survival outcomes in HNSCC patients treated with cetuximab-based chemotherapy. Methods Baseline gene, serum and tumor expression of interleukin-1 (IL-1) ligands were analyzed from The Cancer Genome Atlas (TCGA) database or clinical trials of cetuximab-based therapies and interrogated for associations with clinical outcome data. Results High tumor gene expression of IL-1β was associated with a more favorable overall survival in cetuximab-treated HNSCC patients but not in non-cetuximab-treated patients. In HNSCC patients treated with cetuximab-based chemotherapy, higher gene and circulating levels of IL-1α and IL-1β were correlated with a more favorable progression free survival compared to patients with low or undetectable levels of IL-1 ligands. Conclusions These findings suggest that IL-1 ligands may function as predictive biomarkers for tumor response to cetuximab-based chemotherapy in HNSCC patients and warrants further investigation and validation in larger clinical studies.

Human lung adenocarcinoma (LUAD) in current or former smokers exhibits a high tumor mutational burden (TMB) and distinct mutational signatures. Syngeneic mouse models of clinically relevant smoking-related LUAD are lacking. We established and characterized a tobacco-associated, transplantable murine LUAD cell line, designated FVBW-17, from a LUAD induced by the tobacco carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone in the FVB/N mouse strain. Whole-exome sequencing of FVBW-17 cells identified tobacco-associated KrasG12D and Trp53 mutations and a similar mutation profile to that of classic alkylating agents with a TMB greater than 500. FVBW-17 cells transplanted subcutaneously, via tail vein, and orthotopically generated tumors that were histologically similar to human LUAD in FVB/N mice. FVBW-17 tumors expressed programmed death ligand 1 (PD-L1), were infiltrated with CD8+ T cells, and were responsive to anti–PD-L1 therapy. FVBW-17 cells were also engineered to express green fluorescent protein and luciferase to facilitate detection and quantification of tumor growth. Distant metastases to lung, spleen, liver, and kidney were observed from subcutaneously transplanted tumors. This potentially novel cell line is a robust representation of human smoking-related LUAD biology and provides a much needed preclinical model in which to test promising new agents and combinations, including immune-based therapies., We established and characterized a clinically relevant tobacco carcinogen-induced lung adeno-carcinoma syngeneic cell line that will be useful for preclinical evaluation of immune-oncology agents.

Background: The mesenchymal-epidermal transition (cMET/MET) tyrosine kinase receptor and its ligand, the hepatocyte growth factor (HGF) are overexpressed in a large percentage of non-small cell lung cancers (NSCLCs) and MET mutation and/or amplification leads to oncogene addiction in small subset of NSCLC. In addition, MET amplification is an established mechanism of resistance to EGFR and other oncogenic targeted TKIs. Several MET-inhibitors have been developed and two MET TKIs have been approved for MET exon 14 skipping mutant NSCLC and have shown activity against MET amplified NSCLC. However, long-term efficacy of MET TKIs is limited as acquired resistance is inevitable. HGF overexpression has been identified as one of the mechanisms of resistance to MET TKIs in MET-altered NSCLC, but the mechanism(s) by which the HGF-MET pathway causes resistance are poorly understood. Here, we investigated the requirement of the EMT-transcription factor, TWIST1 in HGF-mediated resistance to MET TKIs and the role of TWIST1 in de-novo and acquired resistance to MET TKIs. Methods: TWIST1 expression was measured in wild type and TWIST1 over expressing cell lines in presence and absence of HGF. TWIST1 was inhibited with shRNA and harmine. Apoptosis was assessed via immunoblotting and cleaved caspase 3 staining. We utilized MET altered (mutant/amplified) NSCLC cell lines, patient derived xenografts and a novel transgenic mouse model of Hgf, Twist1 overexpressing lung cancer to evaluate TWIST1 as a driver of MET TKI resistance. Results: We found that HGF induced TWIST1 expression and MET TKI treatment decreased TWIST1 expression through a post-translational mechanism. Re-expression of TWIST1 led to MET TKI resistance in MET amplified or MET mutant cell lines. Conversely, genetic and pharmacological inhibition of TWIST1 sensitizes to MET inhibition and overcame HGF-mediated MET TKI resistance and MET amplification mediated EGFR TKI resistance in vitro and in vivo. Furthermore, the role of TWIST1 in HGF/MET lung tumorigenesis was evaluated both in human NSCLC xenografts and an Hgf-driven NSCLC mouse model. Genetic inhibition of TWIST1 in MET mutant or amplified cell lines prevented tumor growth in vivo. Furthermore, TWIST cooperated with Hgf in a CCSP-Hgf (CH) mice model that constitutively overexpresses Hgf in the lung and develops NSCLC after treatment with the tobacco carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) was utilized. We demonstrated that the Twist1 overexpressing CHT (CCSP-rtTA/CCSP-Hgf/Twist1-tetO-luc) mice developed significantly larger and more aggressive tumors as compared to CH and CCSP-rtTA/Twist1-tetO-luc (CT) mice and that continued TWIST1 expression was required for these tumors. Conclusions: Our findings suggest that targeting TWIST1 may be an effective therapeutic strategy to overcome HGF-MET-driven resistance in MET-driven NSCLC. Citation Format: Vinod Kumar, Zachary A. Yochum, Princey Devadassan, Eric Huang, Ethan Miller, Vasavi Ayyala, Laura P. Stabile, Timothy F. Burns, Purva H. Rumde. TWIST1 inhibition overcomes resistance to tyrosine kinase inhibitors in MET driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1091.

Tumor immune escape is now a hallmark of cancer development, and therapies targeting these pathways have emerged as standard of care. Specifically, immune checkpoint signal blockade offers durable responses and increased overall survival. However, the majority of cancer patients still do not respond to checkpoint blockade immune therapy leading to an unmet need in tumor immunology research. Sex-based differences have been noted in the use of cancer immunotherapy suggesting that sex hormones such as estrogen may play an important role in tumor immune regulation. Estrogen signaling already has a known role in autoimmunity, and the estrogen receptor can be expressed across multiple immune cell populations and effect their regulation. While it has been well established that tumor cells such as ovarian carcinoma, breast carcinoma, and even lung carcinoma can be regulated by estrogen, research into the role of estrogen in the regulation of tumor-associated immune cells is still emerging. In this chapter, we discuss the role of estrogen in the tumor immune microenvironment and the possible immunotherapeutic implications of targeting estrogen in cancer patients.

Cetuximab, an anti-EGFR monoclonal antibody (mAb), is approved for advanced head and neck squamous cell carcinoma (HNSCC) but benefits a minority. An established tumor-intrinsic resistance mechanism is cross-talk between the EGFR and hepatocyte growth factor (HGF)/cMet pathways. Dual pathway inhibition may overcome cetuximab resistance. This Phase I study evaluated the combination of cetuximab and ficlatuzumab, an anti-HGF mAb, in patients with recurrent/metastatic HNSCC. The primary objective was to establish the recommended Phase II dose (RP2D). Secondary objectives included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Mechanistic tumor-intrinsic and immune biomarkers were explored. Thirteen patients enrolled with no dose-limiting toxicities observed at any dose tier. Three evaluable patients were treated at Tier 1 and nine at Tier 2, which was determined to be the RP2D (cetuximab 500 mg/m2 and ficlatuzumab 20 mg/kg every 2 weeks). Median PFS and OS were 5.4 (90% CI = 1.9&ndash, 11.4) and 8.9 (90% CI = 2.7&ndash, 15.2) months, respectively, with a confirmed ORR of 2 of 12 (17%, 90% CI = 6&ndash, 40%). High circulating soluble cMet levels correlated with poor survival. An increase in peripheral T cells, particularly the CD8+ subset, was associated with treatment response whereas progression was associated with expansion of a distinct myeloid population. This well-tolerated combination demonstrated promising activity in cetuximab-resistant, advanced HNSCC.

Lung cancer remains the leading cause of cancer-related deaths worldwide. In order to understand lung cancer biology and evaluate novel therapeutic strategies, preclinical mouse models have been developed that mimic early and advanced-stage lung cancer. Among autochthonous models, carcinogen-induced systems are valuable preclinical tools since tobacco smoking remains the number one risk factor for lung tumor development. Among the several thousand chemicals within cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen with tumorigenic effects described in both mice and humans. Herein, we describe the methodology for inducing lung tumors in mice using the tobacco carcinogen NNK and subsequent lung fixation for quantitative assessment of tumor development and analysis of oncogenic mutations in tumors.

Lung cancer remains the leading cause of cancer-related deaths worldwide. In order to understand lung cancer biology and evaluate novel therapeutic strategies, preclinical mouse models have been developed that mimic early and advanced-stage lung cancer. Among autochthonous models, carcinogen-induced systems are valuable preclinical tools since tobacco smoking remains the number one risk factor for lung tumor development. Among the several thousand chemicals within cigarette smoke, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen with tumorigenic effects described in both mice and humans. Herein, we describe the methodology for inducing lung tumors in mice using the tobacco carcinogen NNK and subsequent lung fixation for quantitative assessment of tumor development and analysis of oncogenic mutations in tumors.

Tumor immune escape is now a hallmark of cancer development, and therapies targeting these pathways have emerged as standard of care. Specifically, immune checkpoint signal blockade offers durable responses and increased overall survival. However, the majority of cancer patients still do not respond to checkpoint blockade immune therapy leading to an unmet need in tumor immunology research. Sex-based differences have been noted in the use of cancer immunotherapy suggesting that sex hormones such as estrogen may play an important role in tumor immune regulation. Estrogen signaling already has a known role in autoimmunity, and the estrogen receptor can be expressed across multiple immune cell populations and effect their regulation. While it has been well established that tumor cells such as ovarian carcinoma, breast carcinoma, and even lung carcinoma can be regulated by estrogen, research into the role of estrogen in the regulation of tumor-associated immune cells is still emerging. In this chapter, we discuss the role of estrogen in the tumor immune microenvironment and the possible immunotherapeutic implications of targeting estrogen in cancer patients.

Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease., Proinflammatory and immunomodulatory events that drive development of K-ras mutant lung adenocarcinoma (LUAD) are poorly understood. Here they develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout mouse model and show a sex-specific role for epithelial Stat3 signaling in K-ras-mutant LUAD.

Background: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Methods: Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts. Results: NSCLC cell lines and patient tumors and exosomes consistently showed significant increases of ADAM10sa relative to their normal, inflammatory and/or benign-tumor controls. Additionally, stage IA-IIB NSCLC primary tumors of patients who died of the disease exhibited greater increases of ADAM10sa than those of patients who survived 5 years following diagnosis and surgery. In contrast, NSCLC cell lines and patient tumors and exosomes did not display increases of ADAM17sa. Conclusions: This study is the first to investigate enzyme-specific proteolytic activities as potential cancer biomarkers. It provides a proof-of-concept that ADAM10sa could be a biomarker for NSCLC early detection and outcome prediction. To ascertain that ADAM10sa is a useful cancer biomarker, further robust clinical validation studies are needed., National Institutes of Health (U.S.) (R01 CA96504)

Purpose: Immunotherapy has revolutionized treatment options for multiple solid tumors, including non-small cell lung cancer (NSCLC). However, these approaches are only effective in 20-30% of NSCLC patients, thus a more comprehensive picture of the immune response in NSCLC is necessary to develop new therapeutic approaches. Current immunotherapies do not consider or target B cells or tertiary lymphoid structures (TLS) despite their predominance in the tumor microenvironment (TME) and key role in the adaptive immune response. We hypothesize that B cells help generate potent, long-term, immune responses against lung tumor cells by educating T cells within TLS and producing tumor-specific antibodies. Methods: Toward our hypothesis, we have evaluated B cells and TLS in healthy donor lungs (smoker and nonsmoker), COPD patients, and NSCLC patients via single cell RNA sequencing, spectral cytometry (Cytek Aurora) and multispectral imaging (Vectra Polaris and Nanostring GeoMx DSP). These cellular and spatial analyses have revealed key differences in B cell infiltration and TLS formation as lung cancer develops. Results: We have observed an increase in mature TLS (with active germinal centers) as patients develop cancer and have identified key genes that govern TLS heterogeneity in the TME. Further, in evaluating the ex vivo function of B cells from patient tumors via antigen presentation and antibody production assays, we have observed differential function of B cells that correlates with B cell activation (increased HLADR, CD40, CD69). Conclusions: Our studies will provide (a) biomarkers to predict lung cancer progression, (b) targets for early immunotherapeutic intervention in COPD patients that might trigger better anti-tumor immunity as patients develop lung cancer, and (c) immunotherapeutic targets in patients with already established NSCLC. Citation Format: Tullia Bruno, Dongyan Liu, Sheryl Kunning, Riyue Bao, Laura Stabile, Ashwin Somasundaram. Targeting B Cells and Tertiary Lymphoid Structures in Lung Cancer Progression [abstract]. In: Proceedings of the 9th Annual Symposium on Global Cancer Research; Global Cancer Research and Control: Looking Back and Charting a Path Forward; 2021 Mar 10-11. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2021;30(7 Suppl):Abstract nr 73.

Lung cancer has the highest incidence of metastases to the brain, with up to 40% of non-small cell lung cancer (NSCLC) patients developing brain metastases (BM). There is a critical need to develop novel treatments to effectively prevent and treat NSCLC BM. MET is a receptor tyrosine kinase that upon binding hepatocyte growth factor (HGF), mediates proliferation, epithelial-mesenchymal transition (EMT), invasion, angiogenesis and metastasis. Recent studies have suggested that the MET pathway may be a significant determinant of metastatic potential to the brain. We evaluated 125 lung adenocarcinoma (LUAD) BM and 477 primary LUAD for MET amplification (amp) by FISH (MET/CEP > 2) as well as other molecular alterations using targeted next-generation sequencing in a subset of LUAD BM (N=74) and primary LUAD (N=171) samples, including 13 paired primary and brain sets. We identified a significant enrichment of MET amp in LUAD BM (19%) compared to primary LUAD (3%; p5) was present in 6.5% of BM compared to 1.3% of primary LUAD cases (p=0.0006). In matched samples, BM-specific MET amp was observed. Non-exon 14 skipping MET activating mutations were also significantly more frequent in LUAD BM (22%) compared to primary LUAD (12%; p=0.05), as well as TP53, KRAS, SMAD4, APC, RB1, RET, ABL1, ALK, and VHL variants (adj. p values Citation Format: Timothy F. Burns, Sanja Dacic, Maria A. Velez, Ashwin Somasundaram, Saveri Bhattacharya, Anish Chakka, Zachary A. Yochum, Jingxiao Jin, Ethan Miller, Brenda F. Kurland, Riyue Bao, Danielle P. Normolle, Sameer Agnihotri, Uma R. Chandran, Laura P. Stabile. MET alterations are enriched in lung adenocarcinoma brain metastases and define a distinct molecular and transcriptomic subtype [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 2218.

Vascular endothelial growth factor down-regulates microRNA-1 (miR-1) in the lung endothelium, and endothelial cells play a critical role in tumor progression and angiogenesis.To examine the clinical significance of miR-1 in non-small cell lung cancer (NSCLC) and its specific role in tumor endothelium.miR-1 levels were measured by Taqman assay. Endothelial cells were isolated by magnetic sorting. We used vascular endothelial cadherin promoter to create a vascular-specific miR-1 lentiviral vector and an inducible transgenic mouse. KRASIn two cohorts of patients with NSCLC, miR-1 levels were lower in tumors than the cancer-free tissue. Tumor miR-1 levels correlated with the overall survival of patients with NSCLC. miR-1 levels were also lower in endothelial cells isolated from NSCLC tumors and tumor-bearing lungs of KP mouse model. We examined the significance of lower miR-1 levels by testing the effects of vascular-specific miR-1 overexpression. Vector-mediated delivery or transgenic overexpression of miR-1 in endothelial cells decreased tumor burden in KP mice, reduced the growth and vascularity of Lewis lung carcinoma xenografts, and decreased tracheal angiogenesis in vascular endothelial growth factor transgenic mice. In endothelial cells, miR-1 level was regulated through phosphoinositide 3-kinase and specifically controlled proliferation, de novo DNA synthesis, and ERK1/2 activation. Myeloproliferative leukemia oncogene was targeted by miR-1 in the lung endothelium and regulated tumor growth and angiogenesis.Endothelial miR-1 is down-regulated in NSCLC tumors and controls tumor progression and angiogenesis.

BACKGROUND Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non–small cell lung cancer. METHODS In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways. RESULTS Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08). CONCLUSIONS Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936–44. © 2017 American Cancer Society.

About 76% of patients with lung adenocarcinoma harbor activating mutations in the receptor tyrosine kinase (RTK)/RAS/RAF pathways, leading to aberrant activation of the mitogen‐activated protein kinase (MAPK) pathways particularly the MAPK/ERK pathway. However, many lung adenocarcinomas lacking these genomic mutations also display significant MAPK pathway activation, suggesting that additional MAPK pathway alterations remain undetected. This study has identified serine/threonine kinase mitogen‐activated protein 4 kinase 4 (MAP4K4) as a novel positive regulator of MAPK/ERK signaling in lung adenocarcinoma. The results showed that MAP4K4 was drastically elevated in lung adenocarcinoma independently of KRAS or EGFR mutation status. Knockdown of MAP4K4 inhibited proliferation, anchorage‐independent growth and migration of lung adenocarcinoma cells, and also inhibited human lung adenocarcinoma xenograft growth and metastasis. Mechanistically, we found that MAP4K4 activated ERK through inhibiting protein phosphatase 2 activity. Our results further showed that downregulation of MAP4K4 prevented ERK reactivation in EGFR inhibitor erlotinib‐treated lung adenocarcinoma cells. Together, our findings identify MAP4K4 as a novel MAPK/ERK pathway regulator in lung adenocarcinoma that is required for lung adenocarcinoma maintenance.