EGFR-driven lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

EGFR-Driven Lung Adenocarcinomas Co-opt Alveolar Macrophage Metabolism and Function to Support EGFR Signaling and Growth.

The limited efficacy of currently approved immunotherapies in EGFR-driven lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophage (TA-AM) proliferation, which supports tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases proinflammatory immune responses. These results reveal new therapeutic combinations for immunotherapy-resistant EGFR-mutant LUADs and demonstrate how cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth. SIGNIFICANCE: Alternate strategies harnessing anticancer innate immunity are required for lung cancers with poor response rates to T cell-based immunotherapies. This study identifies a targetable, mutually supportive, metabolic relationship between macrophages and transformed epithelium, which is exploited by tumors to obtain metabolic and immunologic support to sustain proliferation and oncogenic signaling.

Navigational Bronchoscopy versus Computed Tomography-guided Transthoracic Needle Biopsy for the Diagnosis of Indeterminate Lung Nodules: protocol and rationale for the VERITAS multicenter randomized trial.

Background: Lung nodule incidence is increasing. Many nodules require biopsy to discriminate between benign and malignant etiologies. The gold-standard for minimally invasive biopsy, computed tomography-guided transthoracic needle biopsy (CT-TTNB), has never been directly compared to navigational bronchoscopy, a modality which has recently seen rapid technological innovation and is associated with improving diagnostic yield and lower complication rate. Current estimates of the diagnostic utility of both modalities are based largely on non-comparative data with significant risk for selection, referral, and publication biases. Methods: The VERITAS trial (na V igation E ndoscopy to R each Indeterminate lung nodules versus T ransthoracic needle A spiration, a randomized controlled S tudy) is a multicenter, 1:1 randomized, parallel-group trial designed to ascertain whether electromagnetic navigational bronchoscopy with integrated digital tomosynthesis is noninferior to CT-TTNB for the diagnosis of peripheral lung nodules 10-30 mm in diameter with pre-test probability of malignancy of at least 10%. The primary endpoint is diagnostic accuracy through 12 months follow-up. Secondary endpoints include diagnostic yield, complication rate, procedure duration, need for additional invasive diagnostic procedures, and radiation exposure. Discussion: The results of this rigorously designed trial will provide high-quality data regarding the management of lung nodules, a common clinical entity which often represents the earliest and most treatable stage of lung cancer. Several design challenges are described. Notably, all nodules are centrally reviewed by an independent interventional pulmonology and radiology adjudication panel relying on pre-specified exclusions to ensure enrolled nodules are amenable to sampling by both modalities while simultaneously protecting against selection bias favoring either modality. Conservative diagnostic yield and accuracy definitions with pre-specified criteria for what non-malignant findings may be considered diagnostic were chosen to avoid inflation of estimates of diagnostic utility. Trial registration: ClinicalTrials.gov NCT04250194.

A Multicenter, Single-Arm, Prospective Trial Assessing the Diagnostic Yield of Electromagnetic Bronchoscopic and Transthoracic Navigation for Peripheral Pulmonary Nodules.

Rationale: Strict adherence to procedural protocols and diagnostic definitions is critical to understand the efficacy of new technologies. Electromagnetic navigational bronchoscopy (ENB) for lung nodule biopsy has been used for decades without a solid understanding of its efficacy, but offers the opportunity for simultaneous tissue acquisition via electromagnetic navigational transthoracic biopsy (EMN-TTNA) and staging via endobronchial ultrasound (EBUS). Objective: To evaluate the diagnostic yield of EBUS, ENB, and EMN-TTNA during a single procedure using a strict a priori definition of diagnostic yield with central pathology adjudication. Methods: A prospective, single-arm trial was conducted at eight centers enrolling participants with pulmonary nodules (<3 cm; without computed tomography [CT]- and/or positron emission tomography-positive mediastinal lymph nodes) who underwent a staged procedure with same-day CT, EBUS, ENB, and EMN-TTNA. The procedure was staged such that, when a diagnosis had been achieved via rapid on-site pathologic evaluation, the procedure was ended and subsequent biopsy modalities were not attempted. A study finding was diagnostic if an independent pathology core laboratory confirmed malignancy or a definitive benign finding. The primary endpoint was the diagnostic yield of the combination of CT, EBUS, ENB, and EMN-TTNA. Measurements and Main Results: A total of 160 participants at 8 centers with a mean nodule size of 18 ± 6 mm were enrolled. The diagnostic yield of the combined procedure was 59% (94 of 160; 95% confidence interval [CI], 51-66%). Nodule regression was found on same-day CT in 2.5% of cases (4 of 160; 95% CI, 0.69-6.3%), and EBUS confirmed malignancy in 7.1% of cases (11 of 156; 95% CI, 3.6-12%). The yield of ENB alone was 49% (74 of 150; 95% CI, 41-58%), that of EMN-TTNA alone was 27% (8 of 30; 95% CI, 12-46%), and that of ENB plus EMN-TTNA was 53% (79 of 150; 95% CI, 44-61%). Complications included a pneumothorax rate of 10% and a 2% bleeding rate. When EMN-TTNA was performed, the pneumothorax rate was 30%. Conclusions: The diagnostic yield for ENB is 49%, which increases to 59% with the addition of same-day CT, EBUS, and EMN-TTNA, lower than in prior reports in the literature. The high complication rate and low diagnostic yield of EMN-TTNA does not support its routine use. Clinical trial registered with www.clinicaltrials.gov (NCT03338049).

EGFR + lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth.

The limited efficacy of currently approved immunotherapies in EGFR-mutant lung adenocarcinoma (LUAD) underscores the need to better understand mechanisms governing local immunosuppression. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophages (TA-AM) to proliferate and support tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF-PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases T cell effector functions. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how such cancer cells can metabolically co-opt TA-AMs through GM-CSF-PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.

Learning Curve of Real-Time Imaging with C-Arm Based Tomography for Peripheral Lung Nodule Biopsy.

The number of procedures required to attain proficiency with new bronchoscopic biopsy technologies for peripheral pulmonary lesions (PPLs) is uncertain. A prospective, single-center study evaluated learning curves of two operators performing PPL biopsies using a novel, real-time, intraoperative tomographic imaging system in consecutive procedures in adults with CT-detected PPLs. Operators were considered "proficient" when they asked three or fewer questions of the manufacturer's clinical representative with no subsequent navigations in which they asked more than three questions. A total of 31 procedures were performed on 31 patients (Operator 1: 18, Operator 2: 13). Proficiency was achieved after an average of 10 procedures (Operator 1: 12, Operator 2: 8). From the learning curve to the post-learning curve period, the number of questions (median [IQR]: 23 [9.5-41.5] versus 0 [0-1], p < 0.001) and radiation dose (median [IQR]: 19.5 mGy/m2 [1.9-43.5] versus 1.5 mGy/m2 [0.7-3.3], p = 0.05) decreased significantly; procedure time decreased (median [IQR]: 12 min [7-20] versus 8 min [3-15], p = 0.29); and diagnostic yield increased significantly (13/20 cases [65%] to 11/11 cases [100%]), (p = 0.03). Based on this unique, clinically relevant method of assessing learning curve, proficiency with the Body Vision system was achieved at approximately the tenth procedure. These findings require validation in larger, diverse populations.

Tracheostomy for COVID-19 Respiratory Failure: Multidisciplinary, Multicenter Data on Timing, Technique, and Outcomes.

OBJECTIVE: The aim of this study was to assess the outcomes of tracheostomy in patients with COVID-19 respiratory failure. SUMMARY BACKGROUND DATA: Tracheostomy has an essential role in managing COVID-19 patients with respiratory failure who require prolonged mechanical ventilation. However, limited data are available on how tracheostomy affects COVID-19 outcomes, and uncertainty surrounding risk of infectious transmission has led to divergent recommendations and practices. METHODS: It is a multicenter, retrospective study; data were collected on all tracheostomies performed in COVID-19 patients at 7 hospitals in 5 tertiary academic medical systems from February 1, 2020 to September 4, 2020. RESULT: Tracheotomy was performed in 118 patients with median time from intubation to tracheostomy of 22 days (Q1-Q3: 18-25). All tracheostomies were performed employing measures to minimize aerosol generation, 78.0% by percutaneous technique, and 95.8% at bedside in negative pressure rooms. Seventy-eight (66.1%) patients were weaned from the ventilator and 18 (15.3%) patients died from causes unrelated to tracheostomy. No major procedural complications occurred. Early tracheostomy (≤14 days) was associated with decreased ventilator days; median ventilator days (Q1-Q3) among patients weaned from the ventilator in the early, middle and late groups were 21 (21-31), 34 (26.5-42), and 37 (32-41) days, respectively with P = 0.030. Compared to surgical tracheostomy, percutaneous technique was associated with faster weaning for patients weaned off the ventilator [median (Q1-Q3): 34 (29-39) vs 39 (34-51) days, P = 0.038]; decreased ventilator-associated pneumonia (58.7% vs 80.8%, P = 0.039); and among patients who were discharged, shorter intensive care unit duration [median (Q1-Q3): 33 (27-42) vs 47 (33-64) days, P = 0.009]; and shorter hospital length of stay [median (Q1-Q3): 46 (33-59) vs 59.5 (48-80) days, P = 0.001]. CONCLUSION: Early, percutaneous tracheostomy was associated with improved outcomes compared to surgical tracheostomy in a multi-institutional series of ventilated patients with COVID-19.

Photodynamic therapy for the treatment of tracheobronchial papillomatosis: A multicenter experience.

OBJECTIVE: Recurrent respiratory papillomatosis (RRP) causes mucosal wart-like growths of the upper aerodigestive tract, which can undergo malignant transformation. These tumors are difficult to treat, often requiring repeated debridement, which can be associated with high morbidity. Photodynamic therapy (PDT) uses a photosensitizing medication and a topically applied light source to treat early stage endobronchial lung cancer. Most data on the use of PDT in RRP pertain to laryngeal disease. Our objective was to evaluate the effectiveness of PDT in treating RRP involving the lower respiratory tract. METHODS: We performed a retrospective multicenter review of adult patients who had a diagnosis of RRP involving the lower airways. We documented details of their disease, treatments, and outcomes. RESULTS: Eight patients underwent PDT for ten RRP lesions. Lesions were located in the trachea and more distal airways. Pathology showed malignant conversion to squamous cell carcinoma in half of the cases. All patient underwent debulking and multimodal treatment concurrently with PDT. Treatment was successful in seven patients with improvement in luminal size. Duration of disease-free recurrence ranged from 4 to 33 months. Five of eight patients have sustained ongoing treatment effect, ranging from 10 to 33 months. Most patient had improved quality of life (83 %) and a reduction in interventions (87 %) after PDT. Complications were minimal. CONCLUSION: PDT can be a safe and effective tool when treating RRP of the lower respiratory tract, including lesions with malignant transformation. A multimodal treatment approach is associated with improved outcomes. Further prospective studies are needed to fully determine its effectiveness.

Clinical and radiographic predictors of successful therapeutic bronchoscopy for the relief of malignant central airway obstruction.

BACKGROUND: Malignant central airway obstruction (CAO) occurs in approximately 20-30% of patients with lung cancer and is associated with debilitating symptoms and poor prognosis. Multimodality therapeutic bronchoscopy can relieve malignant CAO, though carries risk. Evidence to guide clinicians regarding which patients may benefit from such interventions is sparse. We aimed to assess the clinical and radiographic predictors associated with therapeutic bronchoscopy success in relieving malignant CAO. METHODS: We reviewed all cases of therapeutic bronchoscopy performed for malignant CAO at our institution from January 2010-February 2017. Therapeutic bronchoscopy success was defined as establishing airway patency of > 50%. Patient demographics and baseline characteristics, oncology history, degree of airway obstruction, procedural interventions, and complications were compared between successful and unsuccessful groups. Univariate and multivariate logistic regression identified the significant clinical and radiographic predictors for therapeutic success. The corresponding simple and conditional odds ratio were calculated. A time-to-event analysis with Kaplan-Meier plots was performed to estimate overall survival. RESULTS: During the study period, 301 therapeutic bronchoscopies were performed; 44 (14.6%) were considered unsuccessful. Factors associated with success included never vs current smoking status (OR 5.36, 95% CI:1.45-19.74, p = 0.010), patent distal airway on CT imaging (OR 15.11, 95% CI:2.98-45.83, p < 0.0001) and patent distal airway visualized during bronchoscopy (OR 10.77, 95% CI:3.63-31.95, p < 0.001) in univariate analysis. Along with patent distal airway on CT imaging, increased time from radiographic finding to therapeutic bronchoscopy was associated with lower odds of success in multivariate analysis (OR 0.96, 95% CI:0.92-1.00, p = 0.048). Median survival was longer in the successful group (10.2 months, 95% CI:4.8-20.2) compared to the unsuccessful group (6.1 months, 95% CI:2.1-10.8, log rank p = 0.015). CONCLUSIONS: Predictors associated with successful therapeutic bronchoscopy for malignant CAO include distal patent airway visualized on CT scan and during bronchoscopy. Odds of success are higher in non-smokers, and with decreased time from radiographic finding of CAO to intervention.

Management of Lung Transplant Bronchial Stenosis With Mitomycin C.

BACKGROUND: Bronchial stenosis is a significant source of morbidity among lung transplant recipients, with etiologies including infection and ischemia of the airways. Current management with balloon bronchoplasty and stents is imperfect and a subset of patients requires multiple procedures to maintain airway patency. Mitomycin C (MMC) has been utilized for its antifibrotic properties in nonmalignant tracheobronchial stenosis but its application is not well studied in post-lung transplant stenosis. We performed this study to assess if MMC application decreases the need for repeated balloon bronchoplasty in lung transplant-related airway stenosis. METHODS: This is a retrospective cohort study of all lung transplant recipients who developed airway stenosis and who were treated with MMC over 4 years. MMC was injected submucosally into the stenotic airway. We compared the rate of bronchoscopic dilation at intervals of 3 and 6 months before and after MMC therapy. RESULTS: Eleven lung transplant recipients, with airway stenosis were included in our study, who required recurrent balloon dilation, despite airway stents in place in 73% of these patients. At 3 months after MMC treatment the median number of dilations decreased from 3 to 1 (P=0.023), and at 6 months from 3 to 2 dilations (P=0.004). There was a trend toward improvement in forced expiratory volume in one second and forced vital capacity, although it was not statistically significant. No adverse events related to MMC therapy was observed CONCLUSION:: Application of MMC is safe and is associated with a reduction in frequency of bronchoscopic balloon dilation in patients with post-lung transplant airway stenosis.

Yield of Malignant Pleural Effusion for Detection of Oncogenic Driver Mutations in Lung Adenocarcinoma.

BACKGROUND: Pleural fluid can be used to assess targetable mutations in patients with lung adenocarcinoma. The primary objective of this study was to assess the yield of pleural fluid cytology for targetable oncogenic mutations (EGFR, KRAS, BRAF, ALK, and ROS1 gene rearrangements). We also assessed pleural fluid volume necessary for molecular testing. METHODS: Retrospective review was performed of 134 consecutive patients with lung adenocarcinoma associated malignant pleural effusions. EGFR and KRAS testing was done using PCR amplification followed by DNA sequencing, or next generation sequencing in more recent cases that included BRAF assessment. Fluorescence in situ hybridization employing break-apart probes was used to test for ALK and ROS1 rearrangements. RESULTS: Mutation analysis on pleural fluid cell-block was performed on 56 patients. It was adequate for complete analysis ordered including EGFR, KRAS, BRAF, ALK, and ROS1 rearrangements on 40 (71.4%) samples. For individual mutations, EGFR testing was possible in 38 of 49 (77.6%); KRAS 22 of 28 (78.6%); BRAF 10 of 13 (76.9%), ALK gene rearrangement 42 of 51 (82.4%) and ROS1 gene rearrangement in 21 of 28 (75%) pleural fluid specimens. The analysis was satisfactory in 13 of 19 (68.4%) samples with ≤100 mL versus 27 of 37 (72.9%) with >100 mL of fluid tested (P-value=0.7). CONCLUSION: Genetic mutation analysis can be performed on malignant pleural effusions secondary to lung adenocarcinoma, independent of fluid volume.

Needle-based Optical Coherence Tomography to Guide Transbronchial Lymph Node Biopsy.

BACKGROUND: Transbronchial needle aspiration (TBNA), often used to sample lymph nodes for lung cancer staging, is subject to sampling error even when performed with endobronchial ultrasound. Optical coherence tomography (OCT) is a high-resolution imaging modality that rapidly generates helical cross-sectional images. We aim to determine if needle-based OCT can provide microstructural information in lymph nodes that may be used to guide TBNA, and improve sampling error. METHODS: We performed ex vivo needle-based OCT on thoracic lymph nodes from patients with and without known lung cancer. OCT imaging features were compared against matched histology. RESULTS: OCT imaging was performed in 26 thoracic lymph nodes, including 6 lymph nodes containing metastatic carcinoma. OCT visualized lymphoid follicles, adipose tissue, pigment-laden histiocytes, and blood vessels. OCT features of metastatic carcinoma were distinct from benign lymph nodes, with microarchitectural features that reflected the morphology of the carcinoma subtype. OCT was also able to distinguish lymph node from adjacent airway wall. CONCLUSIONS: Our results demonstrate that OCT provides critical microstructural information that may be useful to guide TBNA lymph node sampling, as a complement to endobronchial ultrasound. In vivo studies are needed to further evaluate the clinical utility of OCT in thoracic lymph node assessment.

Surface tension-assisted additive manufacturing.

The proliferation of computer-aided design and additive manufacturing enables on-demand fabrication of complex, three-dimensional structures. However, combining the versatility of cell-laden hydrogels within the 3D printing process remains a challenge. Herein, we describe a facile and versatile method that integrates polymer networks (including hydrogels) with 3D-printed mechanical supports to fabricate multicomponent (bio)materials. The approach exploits surface tension to coat fenestrated surfaces with suspended liquid films that can be transformed into solid films. The operating parameters for the process are determined using a physical model, and complex geometric structures are successfully fabricated. We engineer, by tailoring the window geometry, scaffolds with anisotropic mechanical properties that compress longitudinally (~30% strain) without damaging the hydrogel coating. Finally, the process is amenable to high cell density encapsulation and co-culture. Viability (>95%) was maintained 28 days after encapsulation. This general approach can generate biocompatible, macroscale devices with structural integrity and anisotropic mechanical properties.

Three-dimensional modeled T-tube design and insertion in a patient with tracheal dehiscence.

A 68-year-old man with recurrent medullary thyroid cancer underwent cervical tracheal resection and reconstruction. His course was complicated by tracheal anastomotic dehiscence, right carotid blowout, and ultimately cervical tracheoplasty with AlloDerm. Given the complex vascular interventions and upper-airway anatomy, a custom-designed Montgomery T-tube was designed for him. Three-dimensional digital reconstruction of his upper airways was obtained from a CT scan. The T-tube was designed and fabricated based on the digital trachea model and was subsequently placed successfully. Follow-up CT scan and bronchoscopy confirmed placement and revealed no granulation tissue at 4 weeks. The patient was discharged to home with the ability to phonate. To our knowledge, this is the first demonstration of three-dimensional modeling of an upper-airway defect with subsequent T-tube design using engineering software. The success of this case demonstrates a possible avenue for personalized airway prosthesis design and manufacturing in the future.

Evaluation of rigid bronchoscopy-guided percutaneous dilational tracheostomy. A pilot study.

RATIONALE: Rigid bronchoscopy-guided (RBG) percutaneous tracheostomy has been used in patients with morbid obesity, prior neck surgery, distorted airway anatomy, and uncorrected coagulopathy where standard percutaneous dilational tracheostomy (PDT) is relatively contraindicated. OBJECTIVES: This study aims to describe a standardized approach to incorporate RBG-PDT in clinical practice. METHODS AND MEASUREMENTS: Retrospective case series of patients who underwent RBG-PDT from 2008 to 2012 at Beth Israel Deaconess Medical Center. Patient medical records were reviewed for demographics, comorbid conditions, American Society of Anesthesiologists classification, indication for tracheostomy, duration of procedure, and periprocedural complications. MAIN RESULTS: A total of 35 patients underwent RBG-PDT, including 24 men, with a mean age of 66 years (±11 yr; range, 42-88 yr). The mean body mass index was 34 kg/m(2). The mean procedure time was 32 (±10) minutes, with a median of 33 minutes. The most common indication for tracheostomy was failure to wean from mechanical ventilation, followed by tracheal stenosis and tracheobronchomalacia. The most common indications for RBG-PDT were complex airway, obesity, and coagulopathy. There were no periprocedural complications of consequence, or mortality associated with the procedure. CONCLUSIONS: RBG-PDT is safe and effective in a population of high-risk patients who are otherwise not considered good candidates for standard PDT.

Postradiofrequency ablation inflammatory pseudotumor associated with pulmonary venoocclusive disease: case report and review of the literature.

Radiofrequency ablation of pulmonary veins is a common therapeutic intervention for atrial fibrillation. Pulmonary vein stenosis and venoocclusive disease are recognized complications, but the spectrum of pathologies postablation have not been previously reviewed. A recent case at our hospital showed a left hilar soft tissue mass in association with superior pulmonary vein stenosis in a patient 4 years postablation. On resection, this proved to be an inflammatory pseudotumor composed of myofibroblasts in an organizing pneumonia-type pattern with adjacent dendriform ossifications. Pulmonary venoocclusive change was a prominent feature. Literature on the histopathology of postradiofrequency ablation complications is limited. The severity of vascular pathology appears to increase with the postablation interval. Although pulmonary vascular changes are the most common late finding, fibroinflammatory changes including pulmonary pseudotumor formation, attributable to thermal injury, should be considered in the differential diagnosis of these cases.

Twist is transcriptionally induced by activation of STAT3 and mediates STAT3 oncogenic function.

To explore the basis of metastasis, we compared the human breast cancer lines MCF-7 and MDA-MB453, which have low invasive ability, with their sublines MCF7-I4 and MDA-MB453-I4 with high invasive ability for gene expression and signaling pathways. We previously showed that the I4 lines had dramatically elevated levels of Twist compared with their parental lines. In this study, we observed significantly increased STAT3 Tyr(705) phosphorylation, but not the STAT3 protein levels, in the I4 lines. Activation of STAT3 by interleukin-6 or expression of activated Src induced Twist expression at protein and mRNA levels. Inhibiting STAT3 by a small molecule inhibitor, JSI-124, STAT3 small hairpin RNAs, or dominant negative STAT3 resulted in significant reduction of Twist protein and mRNA expression. STAT3 directly bound to the second proximal STAT3-binding site on the human Twist promoter and activated its transcriptional activity. Inhibition of STAT3 reduced migration, invasion, and colony formation of the I4 cells. Ectopic expression of Twist significantly rescued those phenotypes. Ten normal and 46 tumor specimens of breast tissues were examined for activation of STAT3 and expression of Twist. There was a strong correlation between Tyr(705) p-STAT3 and Twist level in the late stage tumor tissues. Our results indicate that activated STAT3 transcriptionally induces Twist, which plays an important role in promoting migration, invasion, and anchorage-independent growth. Together with our previous observation that Twist transcriptionally induces AKT2 to mediate Twist-promoted oncogenic functions, we conclude that STAT3, Twist, and AKT2 form a functional signaling axis to regulate pivotal oncogenic properties of cancer cells.

Regulation of cancer cell survival, migration, and invasion by Twist: AKT2 comes to interplay.

Metastasis, the foremost cause of mortality in cancer patients, is increasingly recognized as a coordinated biological process. The multistep process of metastasis posts difficulty in studying its mechanism and molecular basis. Recent works have shown that the basic helix-loop-helix transcriptional factor Twist and the serine/threonine kinase AKT play pivotal roles in tumor development and progression. Our recent study has shown that AKT2 is a transcriptional regulatory target of Twist and acts downstream of Twist to promote cancer cell survival, migration, and invasion. Functional convergence of Twist and AKT2 underscores the importance of this signaling pathway in tumor development and progression and as a potential therapeutic target.