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PDF file - 24K, Differences in lesion number between the males and females of the 40mM cohort.

PDF file - 1.3MB, H/E (A) and IHC staining of adenocarcinoma with CK5/6 (B), p63 (C), TTF-1 (D), and NAPSIN-A (E). magnification: 400X.

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PDF file - 99K, The number of animals in each score category from the behavioral index scoring system in the 4mM and 8mM treatment groups.

PDF file - 152K, weight fluctuation in animals receiving 40mM NTCU. This group was divided into two waves (panels A and B), with differing periods of suspensions (arrows) throughout the study. Weight recovery is observable following treatment suspensions. Cessation of treatment is represented with an arrowhead.

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This shows the efficiency of immuodepletion experiments

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The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

Purpose: Lung adenocarcinoma and lung squamous cell carcinoma (SCC) are the most common non–small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of TGFβ type II receptor (TGFβRII) promotes lung adenocarcinoma and SCC carcinogenesis.Experimental Design: We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinicopathologic parameters. To determine whether sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic KrasG12D to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase.Results: Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of KrasG12D-initiated adenocarcinoma and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion.Conclusions: Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII shows haploid insufficiency suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis. Clin Cancer Res; 18(8); 2173–83. ©2012 AACR.

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These figures show experiments of tumor growth in Factor B-/- mice. They also show gating strategy for flow cytometry and changes in inflammatory cells. Data showing loss of CIITA in cancer cells alters the response to complement depletion.

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Objective Lung cancer screening (LCS) efficacy is highly dependent on adherence to annual screening, but little is known about real-world adherence determinants. We used insurance claims data to examine associations between LCS annual adherence and demographic, comorbidity, health care usage, and geographic factors. Materials and Methods Insurance claims data for all individuals with an LCS low-dose CT scan were obtained from the Colorado All Payer Claims Dataset. Adherence was defined as a second claim for a screening CT 10 to 18 months after the index claim. Cox proportional hazards regression was used to define the relationship between annual adherence and age, gender, insurance type, residence location, outpatient health care usage, and comorbidity burden. Results After exclusions, the final data set consisted of 9,056 records with 3,072 adherent, 3,570 nonadherent, and 2,414 censored (unclassifiable) individuals. Less adherence was associated with ages 55 to 59 (hazard ratio [HR] = 0.80, 99% confidence interval [CI] = 0.67-0.94), 60 to 64 (HR = 0.83, 99% CI = 0.71-0.97), and 75 to 79 (HR = 0.79, 99% CI = 0.65-0.97); rural residence (HR = 0.56, 99% CI = 0.43-0.73); Medicare fee-for-service (HR = 0.45, 99% CI = 0.39-0.51), and Medicaid (HR = 0.50, 99% CI = 0.40-0.62). A significant interaction between outpatient health care usage and comorbidity was also observed. Increased outpatient usage was associated with increased adherence and was most pronounced for individuals without comorbidities. Conclusions This population-based description of LCS adherence determinants provides insight into populations that might benefit from specific interventions targeted toward improving adherence and maximizing LCS benefit. Quantifying population-based adherence rates and understanding factors associated with annual adherence are critical to improving screening adherence and reducing lung cancer death.

Background Lung cancer screening (LCS) implementation is complicated by the Centers for Medicare and Medicaid Services reimbursement requirements of shared decision-making and tobacco cessation counseling. LCS programs can utilize different structures to meet these requirements, but the impact of programmatic structure on provider behavior and screening outcomes is poorly described. Patients and Methods In a retrospective chart review of 624 patients in a hybrid structure, academic LCS program, we compared characteristics and outcomes of primary care provider (PCP)- and specialist-screened patients. We also assessed the impact of the availability of an LCS specialty clinic and best practice advisory (BPA) on PCP ordering patterns using electronic medical record generated reports. Results During the study period of July 1, 2014 through June 30, 2018, 48% of patients were specialist-screened and 52% were PCP-screened; there were no clinically relevant differences in patient characteristics or screening outcomes between these populations. PCPs demonstrate distinct practice patterns when offered the choice of specialist-driven or PCP-driven screening. Increased exposure to a LCS BPA is associated with increased PCP screening orders. The addition of a nurse navigator into the LCS program increased documentation of shared decision-making and tobacco cessation counseling to > 95% and virtually eliminated screening of ineligible patients. Conclusions Systematic interventions including a BPA and nurse navigator are associated with increased screening and improved program quality, as evidenced by reduced screening of ineligible patients, increased lung cancer risk of the screened population, and improved compliance with LCS guidelines. Individual PCPs demonstrate clear preferences regarding LCS that should be considered in program design.

We present a case study for implementing a machine learning algorithm with an incremental value framework in the domain of lung cancer research. Machine learning methods have often been shown to be competitive with prediction models in some domains; however, implementation of these methods is in early development. Often these methods are only directly compared to existing methods; here we present a framework for assessing the value of a machine learning model by assessing the incremental value. We developed a machine learning model to identify and classify lung nodules and assessed the incremental value added to existing risk prediction models. Multiple external datasets were used for validation. We found that our image model, trained on a dataset from The Cancer Imaging Archive (TCIA), improves upon existing models that are restricted to patient characteristics, but it was inconclusive about whether it improves on models that consider nodule features. Another interesting finding is the variable performance on different datasets, suggesting population generalization with machine learning models may be more challenging than is often considered.

Lung cancer screening (LCS) has the potential to reduce the risk of lung cancer death in healthy individuals, but the impact of coexisting chronic illnesses on LCS outcomes has not been well defined. Consideration of the complex relationship between baseline risk of lung cancer, treatment-related harms, and risk of death from competing causes is crucial in determining the balance of benefits and harms of LCS.To summarize evidence, identify knowledge and research gaps, prioritize topics, and propose methods for future research on how best to incorporate comorbidities in making decisions regarding LCS.A multidisciplinary group of international clinicians and researchers reviewed available data on the effects of comorbidities on LCS outcomes, focusing on the juxtaposition of lung cancer risk and competing risks of death, consideration of benefits and risks in patients with chronic obstructive pulmonary disease, communication of risk, and treatment of screen-detected lung cancer.This statement identifies gaps in knowledge regarding how comorbidities and competing causes of death impact outcomes in LCS, and we have developed questions to help guide future research efforts to better inform patient selection, education, and implementation of LCS.There is an urgent need for further research that can help guide clinical decision-making with patients who may not benefit from LCS owing to coexisting chronic illness. This statement establishes a research framework to address essential questions regarding how to incorporate and communicate risks of comorbidities into patient selection and decisions regarding LCS.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3−/− mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5–dependent pathway. Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143–56. ©2017 AACR.

Introduction Low-dose computed tomography screening for lung cancer has a high false-positive rate with frequent discovery of indeterminate pulmonary nodules. Noninvasive biomarkers are needed to reduce false positives and improve risk stratification. A retrospective longitudinal evaluation was performed to assess chromosomal aneusomy in sputum by fluorescence in situ hybridization (CA-FISH) in four nested case-control studies. Methods Receiver operating characteristic analysis resulted in two grouped cohorts: a high-risk cohort (Colorado High-Risk Cohort and Colorado Nodule Cohort [68 case patients and 69 controls]) and a screening cohort (American College of Radiology Imaging Network/National Lung Screening Trial and Pittsburgh Lung Screening Study [97 case patients and 185 controls]). The CA-FISH assay was a four-target DNA panel encompassing the EGFR and v-myc avian myelocytomatosis viral oncogene homolog ( MYC ) genes, and the 5p15 and centromere 6 regions or the fibroblast growth factor 1 gene ( FGFR1 ) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene ( PIK3CA ). A four-category scale (normal, probably normal, probably abnormal, and abnormal) was applied. Sensitivity, specificity, and positive and negative likelihood ratios (LRs) (with 95% confidence intervals [CIs]) were estimated for each cohort. Results Sensitivity and specificity were, respectively, 0.67 (95% CI: 0.55–0.78) and 0.94 (95% CI: 0.85–0.98) for high-risk participants and 0.20 (95% CI: 0.13–0.30) and 0.84 (95% CI: 0.78–0.89) for screening participants. The positive and negative LRs were, respectively, 11.66 (95% CI: 4.44–30.63) and 0.34 (95% CI: 0.24–0.48) for high-risk participants and 1.36 (95% CI: 0.81–2.28) and 0.93 (95% CI: 0.83–1.05) for screening participants. Conclusion The high positive LR of sputum CA-FISH indicates that it could be a useful adjunct to low-dose computed tomography for lung cancer in high-risk settings. For screening, however, its low positive LR limits clinical utility. Prospective assessment of CA-FISH in the incidentally identified indeterminate nodule setting is ongoing in the Colorado Pulmonary Nodule Biomarker Trial.

Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules.We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets.We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules.Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair.

Adenoviral vectors expressing Cre recombinase are commonly used to initiate tumor formation in murine lung cancer models. While these vectors are designed to target genetic recombination to lung epithelial cells, adenoviruses can infect additional cell types that potentially influence tumor development. Our goal was to explore the consequences of adenoviral-mediated alveolar macrophage (AM) transduction in a Kras-initiated lung tumor model. As expected, treatment of animals harboring the KrasLSL-G12D allele and an inducible green fluorescence protein (GFP) tracking allele with an adenoviral vector expressing Cre recombinase under the control of the cytomegalovirus (CMV) promoter (Ad5-CMV-Cre), caused GFP-positive lung adenocarcinomas. Surprisingly, however, up to 70% of the total GFP+ cells were AM, and GFP+ AM could be detected 6 months after tumor initiation, and transduced AM demonstrated Kras activation and increased proliferation. In contrast, recombination was not detected in other immune cell populations and AM recombination could be eliminated by tumor initiation with an adenovirus expressing Cre recombinase under the control of the surfactant protein C (SPC) promoter. In addition, AM isolated from KrasLSL-G12D animals and transduced by Ad5-CMV-Cre ex vivo displayed prolonged survival in vitro and increased the growth of murine lung adenocarcinoma CMT/167 cells when co-injected in an orthotopic flank model. Given the importance of the immune system in tumor development and progression, inadvertent AM transduction by Ad5-CMV-Cre merits careful consideration during lung cancer model selection particularly if studies evaluating the tumor-immune interactions are planned.

Lung adenocarcinoma (AdC) and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes, however, most genetic mouse models of lung cancer produce predominantly, if not exclusively, AdC. Whether this is secondary to targeting mutations to the distal airway cells or to the use of activating Kras mutations that drive AdC formation is unknown. We previously showed that targeting Kras(G12D) activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter induced formation of both lung AdC and SCC. In this study we assessed if targeting phosphatase and tensin homologue (PTEN) deletion to airway basal cells could initiate lung tumor formation or increase lung SCC formation. We found that PTEN deletion is capable of initiating both lung AdC and SCC formation when targeted to basal cells and although PTEN deletion is a weaker tumor initiator than Kras(G12D) with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and displayed more malignant conversion than Kras(G12D) initiated tumors. That PTEN deletion did not increase lung SCC formation compared to Kras(G12D) activation, suggests that the initiating genetic event does not dictate tumor histology when genetic alterations are targeted to a specific cell. These studies also confirm that basal cells of the conducting airway are capable of giving rise to multiple NSCLC tumor types.

The authors report a case of a 79-year-old, previously healthy, gentleman who presented with progressive proximal muscle weakness, petechial rash, weight loss, fatigue and diplopia. Neurologic exam demonstrated bilateral esotropia in the primary position and proximal muscle weakness in both upper and lower extremities bilaterally. Subsequent serum laboratory studies, cerebrospinal fluid analysis and neuroimaging failed to identify a unifying diagnosis. The patient was ultimately diagnosed with intravascular large B cell lymphoma (IVLBCL) via skin biopsy and subsequently underwent a single cycle of chemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) before dying from respiratory failure. This case highlights the importance of entertaining a broad differential diagnosis for non-anatomical distribution of central nervous system (CNS) findings, especially when prior investigations into a structural cause have been unrevealing.

Purpose: Lung adenocarcinoma and lung squamous cell carcinoma (SCC) are the most common non–small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of TGFβ type II receptor (TGFβRII) promotes lung adenocarcinoma and SCC carcinogenesis. Experimental Design: We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinicopathologic parameters. To determine whether sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic KrasG12D to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. Results: Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of KrasG12D-initiated adenocarcinoma and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion. Conclusions: Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII shows haploid insufficiency suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis. Clin Cancer Res; 18(8); 2173–83. ©2012 AACR.

Squamous cell carcinoma (SCC) and premalignant endobronchial lesions have been difficult to study in murine models. In this study, we evaluate the topical N-nitroso-tris-chloroethylurea (NTCU) murine SCC model, determine the extent to which resulting premalignant airway dysplasia develops, discuss clinicopathologic grading criteria in lesion progression, and confirm that immunohistochemical (IHC) staining patterns are consistent with those observed in human endobronchial dysplasia and SCC. Male and female FVB mice were treated biweekly with topical NTCU (4, 8, or 40 mmol/L) or vehicle for 32 weeks. Following sacrifice, squamous cell lesions were enumerated and categorized into the following groups: flat atypia, low-grade dysplasia, high-grade dysplasia, and invasive SCC. The 40 mmol/L NTCU concentration produced the entire spectrum of premalignant dysplasias and squamous cell carcinomas, but was associated with poor survival. Concentrations of 4 and 8 mmol/L NTCU were better tolerated and produced only significant levels of flat atypia. Squamous origin of the range of observed lesions was confirmed with IHC staining for cytokeratin 5/6, p63, thyroid transcription factor-1 (TTF-1), and Napsin-A. This study shows that topical application of high-dose NTCU produces endobronchial premalignant lesions with classic squamous characteristics and should allow for improved preclinical evaluation of potential chemopreventive agents. Cancer Prev Res; 5(2); 283–9. ©2011 AACR.

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.

Chromosomal rearrangements of ALK, RET, ROS1, and NTRK1 collectively represent approximately 10% of oncogenic drivers in lung adenocarcinoma. These rearrangements result in the aberrant expression and constitutive activation of a chimeric fusion kinase, which promotes growth, proliferation and survival of the cancer cell. CRISPR/Cas9 technology can now be employed to generate these chromosomal rearrangements both in vitro and in vivo. sgRNAs can be designed to target the Cas9 endonuclease to introns within the 5′ and 3′ gene partners of the rearrangement. A unique advantage of this approach is that it is compatible with immunocompetent mouse models. We have developed a novel in vitro screening strategy to identify sgRNAs that successfully generated the intended rearrangements. Ba/F3 cells are a murine B-cell cell line whose proliferation is normally dependent on the exogenous addition of the cytokine interleukin 3 (IL3), but can be rendered IL3 independent if an oncogenic alteration is introduced. We hypothesized that IL3-independence in Ba/F3 cells could be used as a method to select for cells that had successfully created chromosomal rearrangements leading to oncogenic fusions. This strategy eliminates the need for single cell cloning to confirm that successful CRISPR genome editing has occurred. We transfected Ba/F3 cells with pairs of sgRNAs within the PX330 Cas9 containing plasmid and then withdrew IL3. Cells that became IL3 independent were tested for rearrangements. Using this screening system, we successfully generated Kif5b-Ret, Trim24-Ret, and Tpm3-Ntrk1 rearrangements in Ba/F3 cells. These rearrangements were confirmed using PCR or RT-PCR and sequencing using fusion-specific primers. Additionally, the Ret rearrangements and Ntrk1 rearrangements were far more sensitive to RET and TRK inhibitors, respectively, than parental Ba/F3 cells, further confirming that the Ba/F3 cells' growth was now dependent on signaling from the fusion kinase. RET protein expression was also detected with Western blotting in the two Ret rearranged Ba/F3 cell lines. Our future plans are to introduce adenovirus containing validated sgRNAs into immune-competent mice, and confirm that they generate tumors with the intended rearrangements. Citation Format: Laura Schubert, Anh T. Le, Stephen P. Malkoski, Raphael Nemenoff, Robert C. Doebele. CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B33.

Systems capable of targeting genetic manipulations to keratin-positive airway basal cells are more poorly developed than systems targeting other airway epithelial cell populations and this has likely hindered development of animal models of diseases such as lung squamous cell carcinoma. Although keratin promoter driven-Cre recombinase constructs are potentially useful for targeting these cells, these constructs have substantially higher activity in the skin and oral epithelium than in the airways. We developed a method for delivering RU486, the conditional activator of Cre recombinase progesterone receptor (CrePR) fusion proteins to the lung and then examined the activity of three keratin-driven CrePR constructs in the conducting airways. We also developed a technique for survival bronchioalveolar lavage on non-ventilated animals to examine the effects of the acetone/oil vehicle required to deliver RU486 to the lung. K5CrePR1 and K14CrePR1 constructs differ only in the keratin promoter used to target CrePR1 expression while K5Cre*PR contains a truncated progesterone receptor designed to reduce RU486-independent Cre activity. While all three constructs demonstrate RU486-inducible Cre activity in the conducting airways, both construct activity and tightness of regulation vary considerably. K5Cre*PR is the most tightly regulated Cre driver making it ideal for targeting somatic mutations to the airway epithelia while K5CrePR1 and K14CrePR1 may be better suited to studying diseases of the conducting airways where gene targeting of keratin expressing cells and their derivatives is desired.

Non-small-cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor-suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer; however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC data sets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms, including mutation, homozygous deletion and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4(-/-) mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage, whereas Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4-deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double-strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4-deficient tumors can be exploited by specific therapeutic strategies.

Lung adenocarcinoma (AdC) and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes, however, most genetic mouse models of lung cancer produce predominantly, if not exclusively, AdC. Whether this is secondary to targeting mutations to the distal airway cells or to the use of activating Kras mutations that drive AdC formation is unknown. We previously showed that targeting Kras(G12D) activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter induced formation of both lung AdC and SCC. In this study we assessed if targeting phosphatase and tensin homologue (PTEN) deletion to airway basal cells could initiate lung tumor formation or increase lung SCC formation. We found that PTEN deletion is capable of initiating both lung AdC and SCC formation when targeted to basal cells and although PTEN deletion is a weaker tumor initiator than Kras(G12D) with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and displayed more malignant conversion than Kras(G12D) initiated tumors. That PTEN deletion did not increase lung SCC formation compared to Kras(G12D) activation, suggests that the initiating genetic event does not dictate tumor histology when genetic alterations are targeted to a specific cell. These studies also confirm that basal cells of the conducting airway are capable of giving rise to multiple NSCLC tumor types.

TGFβ signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies.

Exposure to tobacco carcinogens is causally associated with head and neck squamous cell carcinoma (HNSCC), but the underlying molecular mechanisms remain unclear. Here, we reported that AKT is activated at a higher frequency in both HNSCC tumors and the adjacent mucosa from HNSCC patients who are smokers than those from HNSCC patients who are non-smokers. Adding physiologically relevant concentrations of 4-(methylnitrosamino)-1-(3-pyridyl)-1-1butanone (NNK), a major tobacco carcinogen, to normal head and neck epithelial cells and HNSCC cell lines, rapidly and constitutively activated AKT through phosphorylation in a dose- and time-dependent manner. AKT phosphorylation was associated with activation of downstream signaling mediators BAD, MDM2, GSK-3β, mTOR. These alterations correlated with increased proliferation and decreased etoposide-induced apoptosis in NNK-exposed cells. Finally, NNK exposure to mouse head and neck epithelia resulted in epithelial hyperproliferation and reduced apoptosis, which is correlated with AKT activation. Our results suggest that AKT activation is an early event and plays a pivotal role in mediating tobacco-induced HNSCC carcinogenesis.

Tissue-resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin-induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T-cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.

Carboxyl-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor with oncogenic potential. Immunohistochemistry (IHC) staining using human breast cancer tissue arrays revealed that 92% of invasive ductal breast cancer cases have CtBP1-positive staining compared to 4% CtBP1-positive in normal breast tissue. To explore the functional impact of CtBP1 in breast cancer, we examined CtBP1’s transcriptional regulation of known tumor suppressors, breast cancer susceptibility gene 1 (Brca1), and E-cadherin. We found CtBP1 was recruited to the promoter regions of Brca1 and E-cadherin genes in breast cancer cells. Concomitantly, Brca1 loss was detected in 57% and E-cadherin loss was detected in 76% of human invasive ductal breast cancers, and correlated with CtBP1 nuclear staining in these lesions. Importantly, siRNA knock down of CtBP1 restored Brca1 and E-cadherin expression in breast cancer cell lines, implying CtBP1 down-regulates Brca1 and E-cadherin genes in human breast cancer. This study provides evidence that although genetic loss of Brca1 and E-cadherin are infrequent in breast cancer, they are downregulated at the transcriptional level by CtBP1 expression. Thus, CtBP1 activation could be a potential biomarker for breast cancer development.

Head and neck cancer (HNC) is the sixth most common malignancy world-wide, however the survival rate has not improved for the past 20 years. In recent years, the cancer stem cell (CSC) hypothesis has gained ground in several malignancies and there is mounting evidence suggesting CSCs mediate tumor resistance to chemotherapy and radiation therapy. However, the CSC theory is also challenged at least in certain types of cancer. Here we review the progress of CSC studies in HNC, which suggest that HNC conforms to the CSC model. The identified CSC markers and their tumor initiation properties provide a framework for the development of novel therapeutic strategies for HNC.

Carboxyl-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor and metabolic sensory protein, which often represses tumor suppressor genes. Hence, we sought to determine if CtBP1 affects expression of the tumor suppressor Brca1 in head and neck tissue, as down-regulation of Brca1 begins at the early stages of head and neck squamous cell carcinomas (HNSCCs). We found that CtBP1 represses Brca1 transcription by binding to the E2F4 site of the Brca1 promoter. Additionally, the recruitment of CtBP1 to the Brca1 promoter is redox-dependent, i.e., increased at high NADH levels in hypoxic conditions. Further, immunostaining using a human HNSCC tissue array revealed that nuclear CtBP1 staining began to accumulate in hyperplasic lesions and HNSCCs, this staining correlated with Brca1 down-regulation in these lesions. Pharmacological disruption of CtBP1 binding to Brca1 promoter by the antioxidant Tempol, which reduces NADH levels, relieved CtBP1-mediated repression of Brca1, leading to increased DNA repair in HNSCC cells. Since tumor cells are generally hypoxic with increased NADH levels, the dynamic control of Brca1 by a "metabolic switch" found in this study not only provides an important link between tumor metabolism and tumor suppressor expression, but also suggests a potential chemo preventative or therapeutic strategy for HNSCC via blocking NADH-dependent CtBP1 activity at early stages of HNSCC carcinogenesis.

Transforming growth factor beta (TGFβ) is a key regulator of epithelial cell proliferation, immune function and angiogenesis. Because TGFβ signaling maintains epithelial homeostasis, dysregulated TGFβ signaling is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Defective TGFβ signaling in epithelial cells causes hyperproliferation, reduced apoptosis and increased genomic instability, and the compensatory increase in TGFβ production by tumor epithelial cells with TGFβ signaling defects further promotes tumor growth and metastases by increasing angiogenesis and inflammation in tumor stromal cells. Here, we review the mouse models that we used to study TGFβ signaling in HNSCC.

Smad4 is a central mediator of TGF-beta signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4-/- mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4-/- mice exhibited severe inflammation, which was associated with increased expression of TGF-beta1 and activated Smad3. We present what we believe to be the first single gene-knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.