Stephen P. Malkoski

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation

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.

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential.

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. STEM CELLS 2011;29:1283–1293

The Pathology of Bleomycin-Induced Fibrosis Is Associated with Loss of Resident Lung Mesenchymal Stem Cells That Regulate Effector T-cell Proliferation†‡§

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. STEM Cells 2011;29:725–735

TGFβ signaling in head and neck squamous cell carcinoma

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.

A randomized, double-blind, placebo-controlled dose range study of dexmedetomidine as adjunctive therapy for alcohol withdrawal.

Objectives:To evaluate dexmedetomidine as adjunctive therapy to lorazepam for severe alcohol withdrawal. Design:Prospective, randomized, double-blind, placebo-controlled trial. Setting:Single center; medical ICU. Patients:Twenty-four adult patients with a Clinical Institute Withdrawal Assessment score greater than or equal to 15 despite greater than or equal to 16 mg of lorazepam over a 4-hour period. Interventions:Patients received a symptom-triggered Clinical Institute Withdrawal Assessment protocol with lorazepam and were randomized to dexmedetomidine 1.2 &mgr;g/kg/hr (high dose), 0.4 &mgr;g/kg/hr (low dose), or placebo as adjunctive therapy for up to 5 days or resolution of withdrawal symptoms. Measurement and Main Results:High-dose and low-dose groups were combined as a single dexmedetomidine group for primary analysis with secondary analysis exploring a dose-response relationship. The difference in 24-hour lorazepam requirements after versus before study drug was greater in the dexmedetomidine group compared with the placebo group (–56 mg vs –8 mg, p = 0.037). Median differences were similar for high dose and low dose. The 7-day cumulative lorazepam requirements were not statistically different between dexmedetomidine and placebo (159 mg vs 181 mg). Clinical Institute Withdrawal Assessment or Riker sedation-agitation scale scores representing severe agitation (13% vs 25%) or moderate agitation (27% vs 22%) within 24 hours of initiating study drug were similar for dexmedetomidine and placebo groups, respectively. Bradycardia occurred more frequently in the dexmedetomidine group versus placebo group (25% vs 0%, p = not significant), with the majority of bradycardia occurring in the high-dose group (37.5%). Study drug rate adjustments occurred more often in the dexmedetomidine group compared with the placebo group (50% vs 0%, p = 0.02). Neither endotracheal intubation nor seizure occurred in any group while on study drug. Conclusions:Adjunctive dexmedetomidine for severe alcohol withdrawal maintains symptom control and reduces lorazepam exposure in the short term, but not long term, when using a symptom-triggered protocol. Monitoring for bradycardia is needed with dexmedetomidine but the occurrence may be lessened with low dose. Further study is needed to evaluate the clinical impact of dexmedetomidine.

Loss of Transforming Growth Factor Beta Type II Receptor Increases Aggressive Tumor Behavior and Reduces Survival in Lung Adenocarcinoma and Squamous Cell Carcinoma

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.

Transcriptional down-regulation of Brca1 and E-cadherin by CtBP1 in breast cancer†

Carboxyl‐terminal binding protein 1 (CtBP1) is a transcriptional co‐repressor with oncogenic potential. Immunohistochemistry 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 CtBP1s 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 down‐regulated at the transcriptional level by CtBP1 expression. Thus, CtBP1 activation could be a potential biomarker for breast cancer development.

Redox-dependent Brca1 transcriptional regulation by an NADH-sensor CtBP1

C-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 downregulation 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, that is, 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 downregulation 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. As 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 by blocking NADH-dependent CtBP1 activity at early stages of HNSCC carcinogenesis.

A randomized, double-blind pilot study of dexmedetomidine versus midazolam for intensive care unit sedation: patient recall of their experiences and short-term psychological outcomes.

Introduction: Sedation with dexmedetomidine may facilitate ventilator liberation and limit the occurrence of delirium. No trial has assessed patient recall or the development of psychological outcomes after dexmedetomidine sedation. This pilot study evaluated whether transitioning benzodiazepine sedation to dexmedetomidine alters patient recall and the incidence of anxiety, depression, or acute stress disorder (ASD). Methods: This investigation was a randomized, double-blind, single-center study. Existing continuous benzodiazepine sedation was converted to dexmedetomidine or midazolam when patients qualified for daily awakenings. Sedation was titrated to achieve Riker sedation agitation scores of 3 to 4. The intensive care unit (ICU) Stressful Experiences Questionnaire, hospital anxiety and depression scale, and the impact of event scale-revised were administered before hospital discharge to assess recall, anxiety, depression, and manifestations of ASD. Results: A total of 11 patients received dexmedetomidine, and 12 patients received midazolam. Median dosing was 0.61 µg/kg/h for 3.5 days for dexmedetomidine and 3.7 mg/h for 3 days for midazolam. Attainment of goal sedation and analgesia was similar; however, more dexmedetomidine patients experienced agitation and pain. The median duration of mechanical ventilation from study drug initiation to extubation was 3.4 days in dexmedetomidine patients and 2.9 days in midazolam patients. Dexmedetomidine patients remembered 18.5 experiences compared with 8.5 in midazolam patients (P = .015). Rates of anxiety and depression were similar. In all, 5 (62.5%) dexmedetomidine patients and 1 (12.5%) midazolam patient manifested ASD (P = .063), and 1 dexmedetomidine patient and 5 midazolam patients developed new-onset delirium (P = .07). Hypotension occurred in 10 (90.9%) dexmedotomidine patients and 6 (50%) midazolam patients (P = .069). Conclusions: Transitioning benzodiazepine sedation to dexmedetomidine when patients qualify for daily awakenings may reduce the development of delirium and facilitate remembrance of ICU experiences but may lead to manifestations of ASD. Monitoring hypotension is required for both the sedatives. Additional comparative studies focusing on the long-term impact of ICU recall and psychological outcomes are needed.

Tobacco-specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces AKT activation in head and neck epithelia

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.