Human Lung Cancer Specimens Research Articles

Rapid full-color serial sectioning tomography with speckle illumination and ultraviolet excitation

Three-dimensional (3D) high-resolution large-volume imaging has remained a challenge. Translational rapid ultraviolet-excited sectioning tomography (TRUST) achieves rapid and cost-effective whole-organ subcellular imaging through iterative optical scanning and mechanical sectioning. However, the axial resolution is limited by the mechanical sectioning thickness or the UV light penetration depth in tissue. Here, assisted with high-and-low-frequency (HiLo) microscopy (HiLoTRUST), the optical sectioning thickness has been reduced from tens of micrometers to ~5.8 µm. In addition, HiLoTRUST has attained a finer mechanical sectioning thickness (10–15 µm) compared to TRUST (50 µm). For high-content imaging as in TRUST, we employed two additional UV light-emitting diodes (LEDs) specifically for uniform illumination. The full-color imaging ability and improved axial resolution of HiLoTRUST have been validated by two-dimensional (2D)/3D imaging of various mouse organs and human lung cancer specimens. HiLoTRUST offers a cost-effective, full-color, and high-resolution 3D imaging approach, showing its great potential in 3D histology applications.

Human lung cancer harbors spatially organized stem-immunity hubs associated with response to immunotherapy.

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens and found an association with beneficial response to PD-1 blockade. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcome. This hub is distinct from mature tertiary lymphoid structures and is enriched for stem-like TCF7+PD-1+CD8+ T cells, activated CCR7+LAMP3+ dendritic cells and CCL19+ fibroblasts as well as chemokines that organize these cells. Within the stem-immunity hub, we find preferential interactions between CXCL10+ macrophages and TCF7-CD8+ T cells as well as between mature regulatory dendritic cells and TCF7+CD4+ and regulatory T cells. These results provide a picture of the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Abstract 1479: FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS

Abstract Introduction: The role of the endoderm lineage transcription factor FOXA2 in autochthonous lung tumors has been elusive though the expression of FOXA2 has been reported in human lung cancers. Here, using GEMM (Genetically Engineered Mouse Models) and human lung cancer cell lines, we delineate the role of FOXA2 in lung cancers. Experimental Procedures: GEMM that carry mutant KRAS or mutant EGFR along with FOXA2 (CCSP-rtTA; tetO-Foxa2; tetO-Kras.G12D or tetO-EGFR.L858R) were developed allowing for lung tumor induction by doxycycline administration. The growth of lung tumors affected by FOXA2 was assessed by microCT and pathologically. FOXA2 downstream genes were also evaluated in vivo by immunohistochemistry of mouse lung sections and in vitro by gain (ectopic lentivirus transduction) and loss (siRNA) of function approaches with human lung cancer cell lines using TaqMan gene expression, RNA-seq, ChIP-seq and CRISPRi analyses. FOXA2 protein expression in human lung cancer specimens was also assessed. Data: FOXA2 suppressed the growth of EGFR-mutant lung tumors while promoting that of KRAS-mutant lung tumors in GEMM. Notably, half of the KRAS-mutant lung tumors presented invasive mucinous adenocarcinoma of the lung (IMA) with the expression of a master mucous transcription factor SPDEF and the two major IMA mucin genes MUC5AC and MUC5B. In vitro, FOXA2 induced the expression of SPDEF, MUC5AC and MUC5B by directly binding to their loci. H3K27ac was introduced at the enhancer region of MUC5AC (most highly expressed IMA gene) by FOXA2 and the expression of MUC5AC initiated by FOXA2 was enhanced by mutant KRAS. FOXA2 was highly expressed in human IMA compared to normal lung. Conclusions: FOXA2 induces IMA in the presence of mutant KRAS by directly regulating the expression of mucous genes. Citation Format: Yutaka Maeda, Koichi Tomoshige, William D. Stuart, Iris M. Fink-Baldauf, Masaoki Ito, Tomoshi Tsuchiya, Takeshi Nagayasu, Tomoki Yamatsuji, Morihito Okada, Takuya Fukazawa, Minzhe Guo. FOXA2 induces invasive mucinous adenocarcinoma of the lung in the presence of mutant KRAS [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 1479.

Ex-vivo exploration of an endobronchial sentinel lymph node procedure in lung cancer for optimizing workflow and evaluating feasibility of novel imaging tools.

Early-stage lung cancer is treated with curative intent by surgery or radiotherapy. However, upstaging is frequently seen after surgery in clinical N0 lung cancer patients, and despite curative intent, 2-year recurrence rates of 9-28% are reported. A sentinel lymph node (SLN) procedure could improve the staging accuracy. We explored the feasibility of performing a navigation bronchoscopy based SLN procedure in human ex-vivo lung cancer specimens to optimize procedural parameters and assess a novel injection tool. Ten lung resection specimens were included and allocated to either peri- or intratumoral injection of a tracer combining 99mTc-nanocolloid and indocyanine green (ICG) while varying the injection volume. A Pioneer Plus catheter with a pre-angulated 24G needle and an ultrasound (US)-element was used to perform real-time US guided transbronchial injections at multiple locations. Thereafter, single photon emission computed tomography/computed tomography (SPECT/CT)-scanning was performed to image injection depots and to assess their location relative to the tumor. An average volume of 0.7 mL (range, 0.3-1.2 mL) with an average activity of 89.5 MBq 99mTc (range, 35.4-188.0 MBq) was injected. Intratumoral injections in non-solid and solid tumors were successful in 100% and 64.3% respectively, while 100% of peritumoral injections in solid tumors were successful. The US-element of the catheter allowed real-time imaging and was able to visualize all tumors and 67.4% of all injections. SPECT/CT-scanning visualized 76.7% of the injection depots. A navigation bronchoscopy mediated SLN procedure seems technically feasible. The Pioneer Plus is a suitable catheter to place tracer depots at multiple intra-/peri-tumoral sites, while receiving real-time feedback on the needle localization in relation to the tumor. The next step of in-vivo injections will determine if tracer drainage to the SLN can also be detected on pre- and per-operative imaging.

Silencing EGFR-upregulated expression of CD55 and CD59 activates the complement system and sensitizes lung cancer to checkpoint blockade.

The complement system is a critical immune component, yet its role in tumor immune evasion and CD8+ T cell activation is not clearly defined. Here, we demonstrate that epidermal growth factor receptor (EGFR)/Wnt signaling induces β-catenin-mediated long noncoding RNA (lncRNA) LINC00973 expression to sponge CD55-targeting miR-216b and CD59-targeting miR-150. The consequently upregulated CD55/CD59 expression suppresses the complement system and cytokine secretion required for CD8+ T cell activation. CD55/CD59-neutralizing antibody treatment or mutation of the LINC00973 promoter activates the complement and CD8+ T cells, inhibiting tumor growth. Importantly, combined anti-CD55/CD59 and anti-programmed death 1 (anti-PD-1) antibody treatments elicit a synergistic tumor-inhibiting effect. In addition, CD55/CD59 levels are inversely correlated with infiltration of M1 macrophages and CD8+ T cells in human lung cancer specimens and predict patient outcome. These findings underscore the critical role of EGFR/Wnt/β-catenin-upregulated CD55/CD59 expression in inhibiting the complement and CD8+ T cell activation for tumor immune evasion and immune checkpoint blockade resistance and identify a potential combination therapy to overcome these effects.

A germline SNP in BRMS1 predisposes patients with lung adenocarcinoma to metastasis and can be ameliorated by targeting c-fos.

About 50% of patients with early-stage, surgically resected lung cancer will develop distant metastasis. There remains an unmet need to identify patients likely to develop recurrence and to design innovative therapies to decrease this risk. Two primary isoforms of BRMS1, v1 and v2, are present in humans. Using next-generation sequencing of BRMS1 on matched human noncancerous lung tissue and non-small cell lung cancer (NSCLC) specimens, we identified single-nucleotide polymorphism (SNP) rs1052566 that results in an A273V mutation of BRMS1v2. This SNP is homozygous (BRMS1v2A273V/A273V) in 8% of the population and correlates with aggressive biology in lung adenocarcinoma (LUAD). Mechanistically, we show that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2 and promotes robust cell invasion and metastases by activation of c-fos-mediated gene-specific transcriptional regulation. BRMS1v2 A273V increases cell invasion in vitro and increases metastases in both tail-vein injection xenografts and LUAD patient-derived organoid (PDO) intracardiac injection metastasis in vivo models. Moreover, we show that BRMS1v2 A273V fails to interact with nuclear Src, thereby activating intratumoral c-fos in vitro. Higher c-fos results in up-regulation of CEACAM6, which drives metastases in vitro and in vivo. Using both xenograft and PDO metastasis models, we repurposed T5224 for treatment, a c-fos pharmacologic inhibitor investigated in clinical trials for arthritis, and observed suppression of metastases in BRMS1v2A273V/A273V LUAD in mice. Collectively, we elucidate the mechanism of BRMS1v2A273V/A273V-induced metastases and offer a putative therapeutic strategy for patients with LUAD who have this germline alteration.

Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling.

There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein–protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.

MARCKS cooperates with NKAP to activate NF-kB signaling in smoke-related lung cancer.

Rationale: Cigarette smoking is a major risk factor for lung cancer development and progression; however, the mechanism of how cigarette smoke activates signaling pathways in promoting cancer malignancy remains to be established. Herein, we aimed to determine the contribution of a signaling protein, myristoylated alanine-rich C kinase substrate (MARCKS), in smoke-mediated lung cancer.Methods: We firstly examined the levels of phosphorylated MARCKS (phospho-MARCKS) in smoke-exposed human lung cancer cells and specimens as well as non-human primate airway epithelium. Next, the MARCKS-interactome and its gene networks were identified. We also used genetic and pharmacological approaches to verify the functionality and molecular mechanism of smoke-induced phospho-MARCKS.Results: We observed that MARCKS becomes activated in airway epithelium and lung cancer cells in response to cigarette smoke. Functional proteomics revealed MARCKS protein directly binds to NF-κB-activating protein (NKAP). Following MARCKS phosphorylation at ser159 and ser163, the MARCKS-NKAP interaction was inhibited, leading to the activation of NF-κB signaling. In a screen of two cohorts of lung cancer patients, we confirmed that phospho-MARCKS is positively correlated with phospho-NF-κB (phospho-p65), and poor survival. Surprisingly, smoke-induced phospho-MARCKS upregulated the expression of pro-inflammatory cytokines, epithelial-mesenchymal transition, and stem-like properties. Conversely, targeting of MARCKS phosphorylation with MPS peptide, a specific MARCKS phosphorylation inhibitor, suppressed smoke-mediated NF-κB signaling activity, pro-inflammatory cytokines expression, aggressiveness and stemness of lung cancer cells.Conclusion: Our results suggest that phospho-MARCKS is a novel NF-kB activator in smoke-mediated lung cancer progression and provide a promising molecular model for developing new anticancer strategies.

Overexpression of CXCR7 accelerates tumor growth and metastasis of lung cancer cells

BackgroundUnder physiological conditions, CXCL12 modulates cell proliferation, survival, angiogenesis, and migration mainly through CXCR4. Interestingly, the newly discovered receptor CXCR7 for CXCL12 is highly expressed in many tumor cells as well as tumor-associated blood vessels, although the level of CXCR7 in normal cells is low. Recently, many studies have suggested that CXCR7 promotes cell growth and metastasis in more than 20 human malignancies, among which lung cancer is the leading cause of cancer-associated deaths worldwide. Thus, the mechanism of CXCR7 in the progression of lung cancer is urgently needed.MethodsFirst, we explored CXCR4 and CXCR7 expression in human lung cancer specimens and cell lines by immunohistochemistry, western blot and flow cytometry. Then, we chose the human lung adenocarcinoma cell line A549 that stably overexpressed CXCR7 through the way of lentivirus-mediated transduction. Next, “wound healing” assay and transwell assay were applied to compare the cell migration and invasion ability, and stripe assay was used to evaluate the cell polarization. Last, our team established a mouse xenograft model of human lung cancer and monitored tumor proliferation and metastasis by firefly luciferase bioluminescence imaging in SCID/Beige mice.ResultsIn clinical lung cancer samples, CXCR7 expression was almost not detected in normal tissue but upregulated in lung tumor tissue, whereas, CXCR4 was highly expressed in both normal and tumor tissues. Furthermore, overexpression of CXCR7 enhanced A549 cell migration and polarization in vitro. Besides, mouse xenograft model of human lung cancer showed that CXCR7 promoted primary lung tumor’s growth and metastasis to the second organ, such as liver or bone marrow in SCID/Beige mice in vivo.ConclusionsThis study describes the multiple functions of CXCR7 in lung cancer. Thus, these results suggest that CXCR7 may be a malignancy marker and may provide a novel target for anticancer therapy.

The Deubiquitinase USP4 Stabilizes Twist1 Protein to Promote Lung Cancer Cell Stemness.

Lung cancer stem cells (CSCs) play a pivotal role in tumor development, drug resistance, metastasis and recurrence of lung cancer. Thus, it is of great importance to study the mechanism by which CSCs are regulated. In this study, we demonstrate that the deubiquitinase USP4 is critically important in promoting lung cancer stemness. Silencing of USP4 leads to reduction of Oct4 and Sox2 expression, decreased CD133+ cell population and inhibition of tumorsphere formation. Conversely, ectopic expression of USP4 significantly enhances lung cancer cell stemness, which is effectively rescued by simultaneous silencing of Twist1. Mechanistically, we identified USP4 as a novel deubiquitinase of Twist1. USP4 binds to, deubiquitinates and stabilizes Twist1 protein. Furthermore, we show that USP4 expression is elevated in human lung cancer specimens and is positively correlated with Twist1 expression. High expression of USP4/Twist1 is associated with poor clinical outcomes of lung cancer patients. Together, this study highlights an important role for USP4 in lung cancer stemness and suggests USP4 as a potential target for lung cancer diagnosis and treatment.

PRAS40 hyperexpression promotes hepatocarcinogenesis

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers, whereas the molecular mechanism remains largely unknown. PRAS40 (encoded by AKT1S1) phosphorylation was increased in human melanoma, prostate cancer and lung cancer specimens, which was considered as the results of Akt activation. However the mechanism in detail and its role in HCC stay elusive.MethodsPRAS40 expression and phosphorylation were analyzed in HCC specimens, and the survival rates of patients were investigated. Functional analyses of PRAS40 in HCC were performed in vivo and in vitro. The miR-124-3p binding sites in PRAS40 were investigated using luciferase assay. MiR-124-3p expression in HCC specimens was examined by In Situ hybridization, and the correlation to PRAS40 level was evaluated.FindingsThe phosphorylation, protein and mRNA levels of PRAS40 were increased significantly in HCC specimens from our cohorts and TCGA database, which was positively correlated to the poor prognosis of HCC patients. Compared to Akt1s1+/+ mice, hepatocarcinogenesis was suppressed in Akt1s1−/− mice, and the activation of Akt was impaired. PRAS40 depletion resulted in the inhibition of HCC cellular proliferation. Tumor suppressor miR-124-3p was found to downregulate PRAS40 expression by targeting its 3′UTR. MiR-124-3p levels were inversely correlated to PRAS40 protein and phosphorylation levels in HCC specimens. The proliferation inhibition by miR-124-3p mimics was partially reversed by exogenous PRAS40 introduction in HCC cells.InterpretationPRAS40 hyperexpression induced by loss of miR-124-3p contributes to PRAS40 hyperphosphorylation and hepatocarcinogenesis. These results could be expected to offer novel clues for understanding hepatocarcinogenesis and developing approaches.

FBXW2 suppresses migration and invasion of lung cancer cells via promoting \u03b2-catenin ubiquitylation and degradation

FBXW2 inhibits proliferation of lung cancer cells by targeting SKP2 for degradation. Whether and how FBXW2 regulates tumor invasion and metastasis is previously unknown. Here, we report that FBXW2 is an E3 ligase for β-catenin. FBXW2 binds to β-catenin upon EGF-AKT1-mediated phosphorylation on Ser552, and promotes its ubiquitylation and degradation. FBXW2 overexpression reduces β-catenin levels and protein half-life, whereas FBXW2 knockdown increases β-catenin levels, protein half-life and transcriptional activity. Functionally, FBXW2 overexpression inhibits migration and invasion by blocking transactivation of MMPs driven by β-catenin, whereas FXBW2 knockdown promotes migration, invasion and metastasis both in vitro and in vivo lung cancer models. In human lung cancer specimens, while FBXW2 levels are inversely correlated with β-catenin levels and lymph-node metastasis, lower FBXW2 coupled with higher β-catenin, predict a worse patient survival. Collectively, our study demonstrates that FBXW2 inhibits tumor migration, invasion and metastasis in lung cancer cells by targeting β-catenin for degradation.

MicroRNA-148b regulates tumor growth of non-small cell lung cancer through targeting MAPK/JNK pathway

BackgroundMicroRNA-148b (miR-148b) has been detected in various types of tumors, and is generally viewed as a tumor suppressor. Our previous study found the decreased expression of miR-148b in human non small cell lung cancer (NSCLC) specimens and cell lines. However, the underlying mechanisms of miR-148b in regulating tumor progression remain unclear.MethodsFirstly animal experiments were performed to verify whether miR-148b could inhibit the tumor growth. Then, the underlying mechanisms were studied by transfecting recombinant plasmids containing a miR-148b mimic or a negative control (NC) mimic (shRNA control) into NSCLC cell lines PC14/B and A549 cells. Tumor cells transfected with unpackaged lentiviral vectors was used as blank control. Cell proliferation capabilities were measured by using CCK-8 kit and colony formation assay. Cell cycle arrest was compared to clarify the mechanism underlying the tumor cell proliferation. Annexin V-FITC Apoptosis Detection kit was applied to investigate the effect of miR-148b on cell apoptosis. Furthermore, western blot analysis were performed to study the targeting pathway.ResultsWe found that over-expression of miR148b could significantly inhibit tumor growth, while knocking down miR148b could obviously promote tumor growth. Further experiment showed that miR-148b inhibited tumor cell proliferation. Besides, over-expression of miR148b decreased the G2/M phase population of the cell cycle by preventing NSCLC cells from entering the mitotic phase and enhanced tumor cell apoptosis. Further western blot analysis indicated that miR148b could inhibit mitogen-activated protein kinase/Jun N-terminal kinase (MAPK/JNK) signaling by decreasing the expression of phosphorylated (p) JNK.ConclusionsThese results demonstrate that miR-148b could inhibit the tumor growth and act as tumor suppressor by inhibiting the proliferation and inducing apoptosis of NSCLC cells by blocking the MAPK/JNK pathway.

Feasibility of Perioperative Micro–Computed Tomography of Human Lung Cancer Specimens: A Pilot Study

Lesion localization during intraoperative frozen section of lung resection specimens can be challenging. Imaging could aid lesion localization while enabling 3-dimensional specimen analysis. To assess the feasibility of integrating micro-computed tomography (micro-CT) into the perioperative evaluation of fresh surgical lung resection specimens. Fresh lung specimens from patients with a presumptive diagnosis of lung cancer were imaged with micro-CT prior to routine histopathologic and molecular analysis. Micro-CT images were assessed to determine image quality, lesion size, and distance from lesion to the nearest surgical margin. Micro-CT measurements were compared to pathologic measurements using Bland-Altman analysis. A total of 22 specimens from 21 patients were analyzed (mean image acquisition time, 13 ± 6 minutes). Histologic quality of imaged specimens was indistinguishable from a control group of nonimaged lung specimens. Artifacts, most commonly from specimen deflation (n = 8), obscured fine detail on micro-CT images of 10 specimens. Micro-CT could successfully localize the target lesion in the other 12 specimens. Distance to the nearest surgical margin was determined in 10 specimens. Agreement of micro-CT with final pathology was good, with a mean difference of -2.8% (limits of agreement -14.5% to 20.0%) for lesion size and -0.5 mm (limits of agreement -4.4 to 3.4 mm) for distance to nearest surgical margin. Micro-CT of fresh surgical lung specimens is feasible and has the potential to evaluate the size and location of lesions within resection specimens, as well as distance to the nearest surgical margin, all without compromising specimen integrity.

Malignant Transformation of Human Bronchial Epithelial Cells Induced by Arsenic through STAT3/miR-301a/SMAD4 Loop

Arsenic is a well-known of human carcinogen and miR-301a is an oncogenic microRNA, which links to oncogenesis, however, little is understood about its contribution to arsenic-induced cellular transformation and tumorigenesis. Here, we investigated the role of miR-301a during arsenic-induced cellular transformation and tumor formation. miR-301a was found to be upregulated during arsenic-induced BEAS-2B transformation and the overexpression of miR-301a was dependent on IL-6/STAT3 signaling. Inhibition of miR-301a leads to reduction of cell proliferation, colony formation and cell migration. By using dual luciferase assay, SMAD4 was confirmed to be a direct target of miR-301a in BEAS-2B cells and upregulation of SMAD4 is involved the restraining cell growth and migration. In addition, reducing of miR-301a expression enhances doxorubicin-induced cellular apoptosis of transformed BEAS-2B through up-regulating SMAD4. Furthermore, we demonstrated that downregulation of miR-301a in BEAS-2B attenuates tumor growth in the xenograft model by targeting SMAD4. Of note, the level of miR-301a expression correlated inversely with SMAD4 expression in clinical specimens of human lung cancer. Our findings ascertain that miR-301a is an oncogenic miRNA, which targets SMAD4 to establish an essential mechanism for arsenic-induced carcinogenesis, IL-6/STAT3/miR-301a/SMAD4 signaling pathways.

In vivo CRISPR screening unveils histone demethylase UTX as an important epigenetic regulator in lung tumorigenesis

Lung cancer is the leading cause of cancer-related death worldwide. Inactivation of tumor suppressor genes (TSGs) promotes lung cancer malignant progression. Here, we take advantage of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated somatic gene knockout in a KrasG12D/+ mouse model to identify bona fide TSGs. From individual knockout of 55 potential TSGs, we identify five genes, including Utx, Ptip, Acp5, Acacb, and Clu, whose knockout significantly promotes lung tumorigenesis. These candidate genes are frequently down-regulated in human lung cancer specimens and significantly associated with survival in patients with lung cancer. Through crossing the conditional Utx knockout allele to the KrasG12D/+ mouse model, we further find that Utx deletion dramatically promotes lung cancer progression. The tumor-promotive effect of Utx knockout in vivo is mainly mediated through an increase of the EZH2 level, which up-regulates the H3K27me3 level. Moreover, the Utx-knockout lung tumors are preferentially sensitive to EZH2 inhibitor treatment. Collectively, our study provides a systematic screening of TSGs in vivo and identifies UTX as an important epigenetic regulator in lung tumorigenesis.

Bone marrow-derived fibrocytes promote stem cell-like properties of lung cancer cells

Cancer stem cells (CSCs) represent a minor population that have clonal tumor initiation and self-renewal capacity and are responsible for tumor initiation, metastasis, and therapeutic resistance. CSCs reside in niches, which are composed of diverse types of stromal cells and extracellular matrix components. These stromal cells regulate CSC-like properties by providing secreted factors or by physical contact. Fibrocytes are differentiated from bone marrow-derived CD14+ monocytes and have features of both macrophages and fibroblasts. Accumulating evidence has suggested that stromal fibrocytes might promote cancer progression. However, the role of fibrocytes in the CSC niches has not been revealed. We herein report that human fibrocytes enhanced the CSC-like properties of lung cancer cells through secreted factors, including osteopontin, CC-chemokine ligand 18, and plasminogen activator inhibitor-1. The PIK3K/AKT pathway was critical for fibrocytes to mediate the CSC-like functions of lung cancer cells. In human lung cancer specimens, the number of tumor-infiltrated fibrocytes was correlated with high expression of CSC-associated protein in cancer cells. These results suggest that fibrocytes may be a novel cell population that regulates the CSC-like properties of lung cancer cells in the CSC niches.

CD38 knockout suppresses tumorigenesis in mice and clonogenic growth of human lung cancer cells.

The ectodomain of the plasma membrane ectoenzyme CD38 functions as both an NAD glycohydrolase and an ADP-ribosyl cyclase by catalyzing, respectively, the conversion of NAD to nicotinamide and ADP-ribose or cyclic ADP-ribose. CD38 is attracting particular attention in cancer therapy. An anti-CD38 monoclonal antibody (daratumumab) was approved for treatment of patients with multiple myeloma. However, the role of CD38 in non-hematological malignancies has not been explored. Previously, we reported that ADP-ribose-acceptor hydrolase (ARH)-1 deficiency in mice was associated with tumor development. In the present study, we found that in wild-type and ARH1-deficient mice deletion of the CD38 gene reduced tumor formation. Significant reductions in tumor number were observed in lymphomas, adenocarcinomas and hemangio/histolytic sarcomas. Consistent with a role for CD38 in tumorigenesis, CRISPR/Cas9-based knockout of CD38 in A549 human adenocarcinoma cells inhibited anchorage-independent cell growth, cell invasion and xenograft growth in nude mice. CD38 mRNA and protein expression were evaluated in human lung cancer cell lines and in human lung cancer specimens. CD38 overexpression in tumor cells was identified in 11 of 27 patient samples. In addition, some human lung cancer cell lines had dramatically higher CD38 mRNA and protein expression than normal cells. Consistent with these observations, search of the Oncomine database showed that some human lung adenocarcinomas had higher CD38 mRNA levels compared to normal lung tissues. In total, our data are consistent with the conclusion that CD38 plays a role in murine and human lung tumorigenesis and that anti-CD38 treatment may have therapeutic potential in lung cancer.

Lung Cancer Subtypes Generate Unique Immune Responses.

Lung cancer, the leading cause of cancer-related deaths worldwide, is a heterogeneous disease comprising multiple histologic subtypes that harbor disparate mutational profiles. Immune-based therapies have shown initial promise in the treatment of lung cancer patients but are limited by low overall response rates. We sought to determine whether the host immune response to lung cancer is dictated, at least in part, by histologic and genetic differences, because such correlations would have important clinical ramifications. Using mouse models of lung cancer, we show that small cell lung cancer (SCLC) and lung adenocarcinoma (ADCA) exhibit unique immune cell composition of the tumor microenvironment. The total leukocyte content was markedly reduced in SCLC compared with lung ADCA, which was validated in human lung cancer specimens. We further identified key differences in immune cell content using three models of lung ADCA driven by mutations in Kras, p53, and Egfr Although Egfr-mutant cancers displayed robust myeloid cell recruitment, they failed to mount a CD8+ immune response. In contrast, Kras-mutant tumors displayed significant expansion of multiple immune cell types, including CD8+ cells, regulatory T cells, IL-17A-producing lymphocytes, and myeloid cells. A human tissue microarray annotated for KRAS and EGFR mutations validated the finding of reduced CD8+ content in human lung ADCA. Taken together, these findings establish a strong foundational knowledge of the immune cell contexture of lung ADCA and SCLC and suggest that molecular and histological traits shape the host immune response to cancer.

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.