Scott Atay

Mithramycin Represses Basal and Cigarette Smoke-Induced Expression of ABCG2 and Inhibits Stem Cell Signaling in Lung and Esophageal Cancer Cells

Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

Aryl Hydrocarbon Receptor-Induced Adrenomedullin Mediates Cigarette Smoke Carcinogenicity in Humans and Mice

Cigarette smoking (CS) is a leading cause of death worldwide. The aryl hydrocarbon receptor (AHR) is partially responsible for tobacco-induced carcinogenesis although the underlying mechanisms involving early effector genes have yet to be determined. Here, we report that adrenomedullin (ADM) significantly contributes to the carcinogenicity of tobacco-activated AHR. CS and AHR activating ligands induced ADM in vitro and in vivo but not in AHR-deficient fibroblasts and mice. Ectopic transfection of AHR rescued ADM expression in AHR(-/-) fibroblasts whereas AHR blockage with siRNA in wild type cells significantly decreased ADM expression. AHR regulates ADM expression through two intronic xenobiotic response elements located close to the start codon in the ADM gene. Using tissue microarrays we showed that ADM and AHR were coupregulated in lung tumor biopsies from smoker patients. Microarray meta-analysis of 304 independent microarray experiments showed that ADM is elevated in smokers and smokers with cancer. In addition, ADM coassociated with a subset of AHR responsive genes and efficiently differentiated patients with lung cancer from nonsmokers. In a novel preclinical model of CS-induced tumor progression, host exposure to CS extracts significantly elevated tumor ADM although systemic treatment with the ADM antagonist NSC16311 efficiently blocked tobacco-induced tumor growth. In conclusion, ADM significantly contributes the carcinogenic effect of AHR and tobacco combustion products. We suggest that therapeutics targeting the AHR/ADM axis may be of clinical relevance in the treatment of tobacco-induced pulmonary malignancies.

Long-term survival outcomes of cancer-directed surgery for malignant pleural mesothelioma: Propensity score matching analysis

Purpose Small observational studies have shown a survival advantage to undergoing cancer-directed surgery for malignant pleural mesothelioma (MPM); however, it is unclear if these results are generalizable. Our purpose was to evaluate survival after treatment of MPM with cancer-directed surgery and to explore the effect surgery interaction with chemotherapy or radiation therapy on survival by using the National Cancer Database. Patients and Methods Patients with microscopically proven MPM were identified within the National Cancer Database (2004 to 2014). Propensity score matching was performed 1:2 and among this cohort, a Cox proportional hazards regression model was used to identify predictors of survival. Median survival was calculated by using the Kaplan-Meier method. Results Of 20,561 patients with MPM, 6,645 were identified in the matched cohort, among whom 2,166 underwent no therapy, 2,015 underwent chemotherapy alone, 850 underwent cancer-directed surgery alone, 988 underwent surgery with chemotherapy, and 274 underwent trimodality therapy. The remaining 352 patients underwent another combination of surgery, radiation, or chemotherapy. Thirty-day and 90-day mortality rates were 6.3% and 15.5%. Cancer-directed surgery, chemotherapy, and radiation therapy were independently associated with improved survival (hazard ratio, 0.77, 0.74, and 0.88, respectively). Stratified analysis revealed that surgery-based multimodality therapy demonstrated an improved survival compared with surgery alone, with no significant difference between surgery-based multimodality therapies; however, the largest estimated effect was when cancer-directed surgery, chemotherapy, and radiation therapy were combined (hazard ratio, 0.52). For patients with the epithelial subtype who underwent trimodality therapy, median survival was extended from 14.5 months to 23.4 months. Conclusion MPM is an aggressive and rapidly fatal disease. Surgery-based multimodality therapy was associated with improved survival and may offer therapeutic benefit among carefully selected patients.

Fluctuations in Spinal Cord Perfusion Pressure: A Harbinger of Delayed Paraplegia After Thoracoabdominal Aortic Repair

Delayed paraplegia (DP) following thoracoabdominal or descending thoracic aortic (TAA/DTA) repair is a dreaded complication. We reviewed our experience with the management of DP using our previously described COPS protocol (blood-pressure stabilization, cerebrospinal-fluid (CSP) draining and O2-delivery). Complete documentation of hourly CSP pressures and detailed hemodynamic variables were available since 2000. A case-control design was used to analyze the extensive hourly data in the perioperative period. Data were analyzed by contingency-tables, t test, and regression analysis, as appropriate. Between 2000 and 2011, we performed 1059 TAA/DTA repairs. Of these, 47 (4.4%) had DP and 31 (2.9%) had immediate neurologic deficit. Postoperatively, renal replacement therapy and drain complications were significantly associated with DP. Variation in systolic blood pressure (SBP) was also highly predictive. Similarly, spinal-cord perfusion pressure (SCPP = SBP ? SP) showed increased risk with greater variability closer to event day (OR 1.3, P = 0.009). Fluctuation of more than 15 mmHg in SBP in a 24-hour period was associated with 3.2-fold increased odds of DP (P = 0.004). In all, 8/47 (17%) made a full recovery, whereas 19 (40%) had partial recovery by discharge. The 30-day mortality was 18/47 (38%) in DP and 7/55 (13%) in controls (P < 0.001). Long-term survival was significantly lower among DP cases (5-year survival of 28% vs. 75%, P < 0.001). DP occurs infrequently and is predictably associated with intraoperative loss of MEP, postoperative renal replacement therapy, drain complications and unstable systolic and spinal-cord perfusion pressures. Increased vigilance is recommended for patients who experience any of these events.

Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells

Purpose: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms. Experimental Design: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, β-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters. Results: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression. Conclusions: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma. Clin Cancer Res; 22(5); 1197–210. ©2015 AACR.

Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas.

Background Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas. Methods Sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability. Results Sequencing analysis revealed one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. Conclusions A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.

Empyema necessitans caused by actinomycosis: A case report

Highlights • Pulmonary actinomycosis often presents as a chronic infection.• Early misdiagnosis as a neoplasm is common.• We present a rare case of rapid progression and actinomycosis empyema necessitans.• Early operative intervention may allow for limited intervention and shortened antibiotic duration.

Perioperative Outcomes of Patients Undergoing Lobectomy on Clopidogrel

BACKGROUND Perioperative management of antiplatelet therapy for patients undergoing pulmonary resection must balance the risk of cardiovascular events with that of hemorrhage. An optimal approach has not been defined in this population. We sought to characterize outcomes of patients undergoing pulmonary lobectomy on antiplatelet therapy with clopidogrel. METHODS A prospective institutional database was retrospectively reviewed to identify all patients undergoing pulmonary lobectomy from 2007 to 2015 who received perioperative clopidogrel. Patients were grouped according to the timing of clopidogrel discontinuation before operation: group I, 5 days or less; group II, 6 to 14 days; and group III, more than 14 days. Analyses were performed to assess the effect of timing of discontinuation on adverse events. RESULTS Sixty-two patients were identified and included in the analysis. The indication for clopidogrel was coronary artery disease in 44 patients (71%), 35 (56%) of whom had prior stent placement. The overall incidences of transfusion and major cardiovascular events were 16% (10 of 62) and 6.5% (4 of 62), respectively. Non-ST elevation myocardial infarctions occurred in 4 patients. Three were in patients with stents, all placed more than 1 year before the operation. There were no significant differences identified in estimated blood loss, operative duration, or perioperative deaths. CONCLUSIONS No significant outcome differences in perioperative cardiovascular or hemorrhagic events were identified among the groups. Although these findings suggest that clopidogrel may be discontinued shortly before the operation with limited risk of bleeding, we also found that holding therapy for more than 14 days preoperatively may be safe in appropriately selected patients.

Compromise Not Required

The optimal management of a large hiatal hernia requires a combination of technical proficiency and sound intraoperative judgment. Even the most experienced of surgeons will find themselves struggling with an ostensibly shortened esophagus or seemingly unreducible hernia. Although technical maneuvers have been described to manage these challenging cases, like all things, they can come at a price. Esophageal lengthening procedures, such as the Collis gastroplasty or one of its many modifications, were developed to provide adequate intra-abdominal esophageal length in cases of a shortened esophagus. Although this technique has been applied successfully with good functional results and low hernia recurrence rates, there are a number of specific complications that may arise. The most feared and morbid being that of a staple line leak, associated abscess, and possible fistula. Obviously, a leak is rare with antireflux surgery, as the standard conduct of an uncomplicated operation does not involve violation of the enteric tract. Additional long-term difficulties that may arise relate to functional acid-secreting gastric mucosa in the neoesophagus, leading to erosion or ulceration. Dysphagia was also thought to be increased with lengthening procedures, although this was not a consistent finding across series. Given the potential for significant adverse outcomes in what is otherwise an operation done for benign disease, some surgeons have all but abandoned esophageal lengthening procedures. The trade-off with this approach is thought to be a suboptimal antireflux operation and higher rates of recurrent hiatal hernia. In this issue of Seminars, we are presented with a study suggesting that perhaps compromise is not required. Banki and colleagues show in their series of laparoscopic repairs of large hiatal hernias (type I > 4 cm, type II-IV) that excellent perioperative outcomes, low incidence of recurrence, and no need for esophageal lengthening procedures all can be achieved. Despite nearly 50% of patients having a type IV hernia, and half of those with a completely intrathoracic stomach, complete reduction with adequateintra-abdominal length was achieved in all cases. Impressively, the rate of recurrence requiring reintervention was 8%, at a median follow-up of 2 years, comparable with other large series using more liberal application of esophageal lengthening. The manner in which the authors were able to achieve such stellar results without esophageal lengthening should not go unnoticed. On the surface, it may appear that the authors were conceding to the possibility of issues in the future by circumventing the challenges associated with esophageal lengthening in the present. Some may argue that in an effort to avoid a lengthening procedure extra effort was exerted in performing an extensive mediastinal dissection of the esophagus to achieve greater length. In reality, though, this effort is hardly a compromise, but rather, perhaps an enhancement to some and a reminder to many surgeons that adequate mobilization of the esophagus should performed routinely with superb technical skill. Perhaps more important is the resolution of symptoms on their follow-up questionnaire. Although 67% of patients presented with *Division of Thoracic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California Jeffrey P. Smith Endowed Chair in Surgery, Division of Thoracic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California

Adjuvant chemotherapy following trimodality therapy for esophageal carcinoma-Is the evidence sufficient?

The preferred management of localized esophageal carcinoma consists of multi-modality therapy (chemotherapy, radiation, and surgery). The ideal timing, treatment sequence, and dose of therapy remain active areas of investigation and controversy. Following publication of favorable outcomes for neoadjuvant chemoradiation prior to definitive resection, tri-modality therapy has become the standard approach in the United States for patients with local/regionally advanced disease (1-5). As the experience with trimodality therapy has grown, adjuvant treatment following completion of all planned therapy has become an area of interest. For those 15–30% of patients with a pathologic complete response (pathCR), the decision to reserve further therapy until the time of recurrence appears straightforward, with no clear evidence of significant clinical benefit for either adjuvant chemotherapy or radiation. However, even for patients with pathCR, recurrence rates remain high with approximately >33% of patients developing distant metastases. In patients with residual disease burden following trimodality therapy, the question of adjuvant chemotherapy becomes more relevant (6-8). This group of patients (pT+ and/or N+) remains at highrisk for both local and/or distant failure. The addition of adjuvant systemic therapy is hoped to provide superior outcomes compared to observation alone (9). Taken together, the evidence would suggest a potential role for adjuvant systemic therapy regardless of pathologic response; the challenge becomes identifying those patients in whom further systemic therapy, with its inherent risks, will offer a relevant clinical benefit.