John Langenfeld

Bone morphogenetic protein 2 stimulation of tumor growth involves the activation of Smad-1/5.

Morphogenetic protein 2 (BMP-2) is normally expressed in the embryo promoting the development of several organs. Aberrant expression of BMP-2 occurs in approximately 98% of lung carcinomas, however, its role in regulating tumor growth is poorly understood. We show that BMP-2 induces Id-1 expression in lung cancer cell lines through its activation of Smad-1/5, which is dependent on cell culture conditions. A549 cells in DMEM/5% FCS BMP-2 activated Smad-1/5 and caused a transient increase in proliferation. In serum-free medium, BMP-2 induced significantly less Smad-1/5 activation and Id-1 expression, and produced significant growth inhibition. The affect of BMP-2 on tumor growth in vivo was substantially more significant. Recombinant BMP-2 coinjected with A549 cells, into nude mice increased proliferation and produced an increase in Id-1 expression. Forced expression of BMP-2 in A549 cells significantly enhanced tumor growth in the lungs following intravenous injection but not of subcutaneous tumors. Tumors in the lung were found to have an activated Smad-1/5 and expressed Id-1. Subcutaneous tumors expressed less activated Smad-1/5 and Id-1 than that of controls. Human lung carcinomas were also found to express an activated Smad-1/5 and Id-1. We provide evidence that BMP-2 promotes tumor growth. This paper highlights that cell culture experiments may not reveal the full biological affects of BMP-2, and its activity varies depending of the local environment.

Bone Morphogenetic Protein-2-Induced Transformation Involves the Activation of Mammalian Target of Rapamycin

Bone morphogenetic protein-2 (BMP-2) is an evolutionary conserved protein that is essential for embryonic development. BMP-2 is highly expressed in ∼98% of human lung carcinomas with little expression in normal lung tissues. BMP-2 has been shown to enhance mobility, invasiveness, and metastasis of cancer cell lines. During development, BMP-2 induces the proto-oncogene phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway to regulate stem cell differentiation. We show that BMP-2 induces the phosphorylation of mTOR in A549 and H1299 lung cancer cell lines, which is attenuated by the PI3K antagonists LY-294002 and wortmannin. p70S6 kinase, which is a direct downstream target of mTOR, is also regulated by BMP-2 in lung cancer cell lines. We find that BMP-2 induces cyclin E in A549 and H1299 cells, which is mediated by the PI3K/mTOR signaling pathway. The regulation of cyclin E by BMP-2 occurs through a Smad 1/5–independent mechanism. Forced expression of BMP-2 in A549 cells (A549/BMP-2) induces transformation as shown by an increase in foci formation. The mTOR antagonist, rapamycin, prevented foci formation of the A549/BMP-2 cells. This study provides evidence that BMP-2-mediated transformation of lung cancer cells involves the activation of the PI3K/mTOR signaling pathway. (Mol Cancer Res 2005;3(12):679–84)

Mycoplasma infection transforms normal lung cells and induces bone morphogenetic protein 2 expression by post-transcriptional mechanisms

Bone morphogenetic protein 2 (BMP2) is an essential growth factor and morphogen, whose pattern and level of expression profoundly influences development and physiology. We present the novel finding that mycoplasma infection induces BMP2 RNA production in six cell lines of diverse types (mesenchymal, epithelial, and myeloid). Mycoplasma infection triggered the expression of mature secreted BMP2 protein in BEAS‐2B cells (immortalized human bronchial epithelial cells), which normally do not express BMP2, and further increased BMP2 production in A549 cells (lung adenocarcinoma cells). Indeed, mycoplasma is as strong an experimental inducer as inflammatory cytokines and retinoic acid. Second, we showed that post‐transcriptional mechanisms including regulation of RNA stability, rather than transcriptional mechanisms, contributed to the increased BMP2 expression in mycoplasma‐infected cells. Furthermore, a novel G‐rich oligonucleotide, AS1411 that binds the post‐transcriptional regulator nucleolin induced BMP2 exclusively in infected cells. Finally, BMP2 stimulated proliferation in BEAS‐2B cells transformed by chronic mycoplasma infection, as demonstrated by treatment with Noggin, a BMP2 antagonist. These findings have important implications regarding the effects of mycoplasma on BMP2‐regulated processes, including cell proliferation, differentiation, and apoptosis. J. Cell. Biochem. 104: 580–594, 2008.

Bone morphogenetic protein type I receptor antagonists decrease growth and induce cell death of lung cancer cell lines.

Bone morphogenetic proteins (BMPs) are highly conserved morphogens that are essential for normal development. BMP-2 is highly expressed in the majority of non-small cell lung carcinomas (NSCLC) but not in normal lung tissue or benign lung tumors. The effects of the BMP signaling cascade on the growth and survival of cancer cells is poorly understood. We show that BMP signaling is basally active in lung cancer cell lines, which can be effectively inhibited with selective antagonists of the BMP type I receptors. Lung cancer cell lines express alk2, alk3, and alk6 and inhibition of a single BMP receptor was not sufficient to decrease signaling. Inhibition of more than one type I receptor was required to decrease BMP signaling in lung cancer cell lines. BMP receptor antagonists and silencing of BMP type I receptors with siRNA induced cell death, inhibited cell growth, and caused a significant decrease in the expression of inhibitor of differentiation (Id1, Id2, and Id3) family members, which are known to regulate cell growth and survival in many types of cancers. BMP receptor antagonists also decreased clonogenic cell growth. Knockdown of Id3 significantly decreased cell growth and induced cell death of lung cancer cells. H1299 cells stably overexpressing Id3 were resistant to growth suppression and induction of cell death induced by the BMP antagonist DMH2. These studies suggest that BMP signaling promotes cell growth and survival of lung cancer cells, which is mediated through its regulation of Id family members. Selective antagonists of the BMP type I receptors represents a potential means to pharmacologically treat NSCLC and other carcinomas with an activated BMP signaling cascade.

Reduction in Tumor Volume by Cone Beam Computed Tomography Predicts Overall Survival in Non-Small Cell Lung Cancer Treated With Chemoradiation Therapy

PURPOSE We sought to evaluate whether tumor response using cone beam computed tomography (CBCT) performed as part of the routine care during chemoradiation therapy (CRT) could forecast the outcome of unresectable, locally advanced, non-small cell lung cancer (NSCLC). METHODS AND MATERIALS We manually delineated primary tumor volumes (TV) of patients with NSCLC who were treated with radical CRT on days 1, 8, 15, 22, 29, 36, and 43 on CBCTs obtained as part of the standard radiation treatment course. Percentage reductions in TV were calculated and then correlated to survival and pattern of recurrence using Cox proportional hazard models. Clinical information including histologic subtype was also considered in the study of such associations. RESULTS We evaluated 38 patients with a median follow-up time of 23.4 months. The median TV reduction was 39.3% (range, 7.3%-69.3%) from day 1 (D1) to day 43 (D43) CBCTs. Overall survival was associated with TV reduction from D1 to D43 (hazard ratio [HR] 0.557, 95% CI 0.39-0.79, P=.0009). For every 10% decrease in TV from D1 to D43, the risk of death decreased by 44.3%. For patients whose TV decreased ≥39.3 or <39.3%, log-rank test demonstrated a separation in survival (P=.02), with median survivals of 31 months versus 10 months, respectively. Neither local recurrence (HR 0.791, 95% CI 0.51-1.23, P=.29), nor distant recurrence (HR 0.78, 95% CI 0.57-1.08, P=.137) correlated with TV decrease from D1 to D43. Histologic subtype showed no impact on our findings. CONCLUSIONS TV reduction as determined by CBCT during CRT as part of routine care predicts post-CRT survival. Such knowledge may justify intensification of RT or application of additional therapies. Assessment of genomic characteristics of these tumors may permit a better understanding of behavior or prediction of therapeutic outcomes.

Small molecule antagonist of the bone morphogenetic protein type I receptors suppresses growth and expression of Id1 and Id3 in lung cancer cells expressing Oct4 or nestin

BackgroundBone morphogenetic proteins (BMP) are embryonic morphogens that are aberrantly expressed in lung cancer. BMPs mediate cell fate decisions and self-renewal of stem cells, through transcription regulation of inhibitor of differentiation protein/DNA binding proteins (Id1-3). Inhibition of BMP signaling decreases growth and induces cell death of lung cancer cells lines by downregulating the expression of Id proteins. It is not known whether the BMP signaling cascade regulates growth and the expression of Id proteins of lung cancer cells expressing the stem cell markers Oct4 and/or nestin.MethodsLung cancer cells expressing Oct4 or nestin were isolated from lung cancer cell lines by stably transfecting the Oct4 promoter or nestin promoter expression vectors that induce expression of the green fluorescent protein reporter.ResultsOur studies suggest that lung cancer cells expressing Oct4 or nestin are different cell populations. Microarray and quantitative RT-PCR demonstrated that the expression of specific stem cell markers were different between isolated Oct4 and nestin cells. Both the Oct4 and nestin populations were more tumorigenic than controls but histologically they were quite different. The isolated Oct4 and nestin cells also responded differently to inhibition of BMP signaling. Blockade of BMP signaling with the BMP receptor antagonist DMH2 caused significant growth inhibition of both the Oct4 and nestin cell populations but only increased cell death in the nestin population. DMH2 also induced the expression of nestin in the Oct4 population but not in the nestin cells. We also show that BMP signaling is an important regulator of Id1 and Id3 in both the Oct4 and nestin cell populations. Furthermore, we show that NeuN is frequently expressed in NSCLC and provide evidence suggesting that Oct4 cells give rise to cancer cells expressing nestin and/or NeuN.ConclusionThese studies show that although biologically different, BMP signaling is growth promoting in cancer cells expressing Oct4 or nestin. Inhibition of BMP signaling decreases expression of Id proteins and suppresses growth of cancer cells expressing Oct4 or Nestin. Small molecule antagonists of the BMP type I receptors represent potential novel drugs to target the population of cancer cells expressing stem cell markers.

Impact of metformin use on survival in locally-advanced, inoperable non-small cell lung cancer treated with definitive chemoradiation.

BACKGROUND We investigated survival outcomes in diabetic patients with non-small cell lung cancer (NSCLC) treated with concurrent metformin and definitive chemoradiation. METHODS This single-institution, retrospective cohort study included 166 patients with NSCLC who were treated definitively with chemoradiation between 1999 and 2013. Of 40 patients who had type II diabetes, 20 (50%) were on metformin, and 20 (50%) were not on metformin. The primary outcome was overall survival (OS), and secondary outcomes included progression-free survival (PFS), locoregional recurrence-free survival (LRRFS) and distant metastasis-free survival (DMFS). Kaplan Meier method and log-rank test were performed in survival analysis. Cox regression was utilized in univariate analysis of potential confounders. RESULTS Median follow-up was 17.0 months. Compared with non-diabetic patients, diabetic patients on metformin demonstrated similar OS (16.3 vs. 14.3 mo, P=0.23), PFS (11.6 vs. 9.7 mo, P=0.26), LRRFS (14.1 vs. 11.9 mo, P=0.78), and DMFS (13.4 vs. 10.0 mo, P=0.69). Compared with diabetic patients not on metformin, diabetic patients on metformin also exhibited similar OS (14.3 vs. 19.2 mo, P=0.18), PFS (19.7 vs. 10.1 mo, P=0.38), LRRFS (11.9 vs. 15.5 mo, P=0.69), and DMFS (10.0 vs. 17.4 mo, P=0.12). Identified negative prognostic factors on included squamous cell histology, lower performance status, higher T stage, and non-caucasian ethnicity. CONCLUSIONS No statistically significant differences in survival or patterns of failure were found among the three cohorts in this small set of patients. No statistically significant differences in survival or patterns of failure were found between the three cohorts in this small set of patients. Though it is possible that metformin use may in fact have no effect on survival in NSCLC patients treated with definitive RT, larger-scale retrospective and prospective studies are implicated for clarification.

Treatment of Diaphragmatic Hernia Occurring After Transhiatal Esophagectomy

BackgroundPostesophagectomy diaphragmatic hernia (DH) is an uncommon problem but an important one to recognize and treat because of the risk of significant complications such as incarceration and strangulation. Diaphragmatic hernia appears to occur more frequently following transhiatal esophagectomy (THE) than after transthoracic procedures, likely because of the enlargement of the diaphragmatic hiatus required to perform THE.MethodsAfter 199 consecutive esophagectomies were performed at Rutgers Robert Wood Johnson University Hospital between January 2000 and June 2013, ten patients were identified with DH; all underwent diaphragmatic hernia repair (DHR). All patients who underwent esophagectomy during this time period were cataloged in a prospectively maintained database that was then retrospectively reviewed. All DH were repaired using a novel biologic plug mesh technique.ResultsTen esophagectomy patients developed DH; nine post-THE and one post-McKeown esophagectomy. One patient was excluded from analysis because of atypical presentation. Demographic data were similar between esophagectomy patients who developed DH and those who did not. Administration of neoadjuvant chemoradiation correlated with development of DH, but did not reach statistical significance. Complications directly related to DHR were few and mostly infectious, including empyema and pneumonia, and were more likely to occur in those who presented with acute obstruction. One patient presented with dysphagia post repair.ConclusionsDiaphragmatic hernia development post esophagectomy is an uncommon complication, but can have devastating results when there is bowel compromise. Repair by plugging the diaphragmatic hiatus with a biologic mesh is a safe and effective method for closing the defect and results in few complications.

Thymolipoma: clues to pathogenesis revealed by cytogenetics.

The pathogenesis of thymolipoma is controversial and unclear despite numerous reports. A case report of thymolipoma with cytogenetic analysis is herein presented. The lesion demonstrated a translocation involving the HMGA2 gene on chromosome 12q15, which is seen in two thirds of solitary lipomas. This finding supports the theory that this case of thymolipoma is a neoplasm of thymic fat.

Outcome-volume relationships and transhiatal esophagectomy: minimizing “failure to rescue”

BackgroundThe objective of this study is to describe the system and technical factors that enabled our moderate size transhiatal esophagectomy program to achieve low mortality rates.MethodsA retrospective chart review was conducted on 200 consecutive patients who underwent transhiatal esophagectomy at Robert Wood Johnson University Hospital. Primary outcomes included operative times, estimated blood loss, frequency and nature of complications, and lengths of stay in the hospital and the intensive care unit.ResultsIn general, surgical outcomes tended to improve over the course of this study. We identified decreased operative time, intra-operative blood loss, frequency of complications, and lengths of intensive care unit and hospital stay as the program matured. Through coordinated actions of the surgical and anesthesia teams, all intraoperative injuries were responded to in an effective, emergent fashion and all but one patient was saved. This resulted in an inhospital and 30-day mortality rate of only 0.5%.ConclusionsOur study suggests that a dual attending approach, focus on avoiding “failure to rescue”, increased volume, and a surgeon driven commitment to quality improvement may lead to low mortality rates after transhiatal esophagectomy.