Che-Chung Yeh

A practical molecular assay to predict survival in resected non-squamous, non-small-cell lung cancer: development and international validation studies.

BACKGROUND The frequent recurrence of early-stage non-small-cell lung cancer (NSCLC) is generally attributable to metastatic disease undetected at complete resection. Management of such patients depends on prognostic staging to identify the individuals most likely to have occult disease. We aimed to develop and validate a practical, reliable assay that improves risk stratification compared with conventional staging. METHODS A 14-gene expression assay that uses quantitative PCR, runs on formalin-fixed paraffin-embedded tissue samples, and differentiates patients with heterogeneous statistical prognoses was developed in a cohort of 361 patients with non-squamous NSCLC resected at the University of California, San Francisco. The assay was then independently validated by the Kaiser Permanente Division of Research in a masked cohort of 433 patients with stage I non-squamous NSCLC resected at Kaiser Permanente Northern California hospitals, and on a cohort of 1006 patients with stage I-III non-squamous NSCLC resected in several leading Chinese cancer centres that are part of the China Clinical Trials Consortium (CCTC). FINDINGS Kaplan-Meier analysis of the Kaiser validation cohort showed 5 year overall survival of 71·4% (95% CI 60·5-80·0) in low-risk, 58·3% (48·9-66·6) in intermediate-risk, and 49·2% (42·2-55·8) in high-risk patients (p(trend)=0·0003). Similar analysis of the CCTC cohort indicated 5 year overall survivals of 74·1% (66·0-80·6) in low-risk, 57·4% (48·3-65·5) in intermediate-risk, and 44·6% (40·2-48·9) in high-risk patients (p(trend)<0·0001). Multivariate analysis in both cohorts indicated that no standard clinical risk factors could account for, or provide, the prognostic information derived from tumour gene expression. The assay improved prognostic accuracy beyond National Comprehensive Cancer Network criteria for stage I high-risk tumours (p<0·0001), and differentiated low-risk, intermediate-risk, and high-risk patients within all disease stages. INTERPRETATION Our practical, quantitative-PCR-based assay reliably identified patients with early-stage non-squamous NSCLC at high risk for mortality after surgical resection. FUNDING UCSF Thoracic Oncology Laboratory and Pinpoint Genomics.

Induction of protein growth factor systems in the ovaries of transgenic mice overexpressing human type 2 lysophosphatidic acid G protein-coupled receptor (LPA2)

The lipid growth factor lysophosphatidic acid (LPA) is produced by ovarian cancer cells in quantities sufficient to attain concentrations of up to 10 μM. An autocrine circuit was demonstrated when ovarian cancer cells, but not normal ovarian surface epithelial cells, were proven to express LPA2 (Edg-4) and LPA3 (Edg-7) G protein-coupled receptors for LPA. Human LPA2 now has been expressed transgenically in C57BL/6 mouse ovaries under direction of the α-inhibin large promoter. Human LPA2 mRNA and protein were detected in all transgenic (TG) mouse ovaries at levels far higher than in other tissues and at least fivefold higher than in cultured lines of human ovarian cancer cells, with the expected sex cord-stromal distribution. Most LPA2 TG ovaries produced significantly higher levels than non-TG ovaries of type A, but not type B, vascular endothelial growth factor (VEGF), isomers of VEGF-A, and urokinase-type plasminogen activator (uPA). Many LPA2 TG ovaries had elevated expression of VEGF receptors 1 and 2, and a depressed level of type 2 PA inhibitor. Thus, the LPA–LPA2 circuit regulates ovarian cells both directly and through increases in protein growth factor systems.

Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse.

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro‐ and anti‐apoptotic MAP kinase signaling in cardiac myocyte (CM) and non‐myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post‐MI remodeling involved dramatic changes in the phosphorylation of both stress‐activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti‐apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p‐ERK localized to fibrotic areas populated primarily by non‐myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non‐myocytes in different regions of the post‐MI myocardium revealed signaling patterns that imply cell type‐specific changes in pro‐ and anti‐apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type‐specific manipulation of p38 and ERK activation. J. Cell. Biochem. 109: 1185–1191, 2010.

Sphingolipid signaling and treatment during remodeling of the uninfarcted ventricular wall after myocardial infarction

The sphingosine kinase (SphK)/sphingosine 1-phosphate (S1P) pathway, known to determine the fate and growth of various cell types, can enhance cardiac myocyte survival in vitro and provide cardioprotection in acute ex vivo heart preparations. However, the relevance of these findings to chronic cardiac pathology has never been demonstrated. We hypothesized that S1P signaling is impaired during chronic remodeling of the uninfarcted ventricle during the evolution of post-myocardial infarction (MI) cardiomyopathy and that a therapeutic enhancement of S1P signaling would ameliorate ventricular dysfunction. SphK expression and activity were measured in the remote, uninfarcted myocardium (RM) of C57Bl/6 mice subjected to coronary artery ligation. The mRNA expression of S1P receptor isoforms was also measured, as was the activation of the downstream S1P receptor mediators. A cardioprotective role for S1P(1) receptor agonism was tested via the administration of the S1P(1)-selective agonist SEW2871 during and after MI. As a result, the expression data suggested that a dramatic reduction in SphK activity in the RM early after MI may reflect a combination of posttranscriptional and posttranslational modulation. SphK activity continued to decline gradually during chronic post-MI remodeling, when S1P(1) receptor mRNA also fell below baseline. The S1P(1)-specific agonism with oral SEW2871 during the first 2-wk after MI reduced apoptosis in the RM and resulted in improved myocardial function, as reflected in the echocardiographic measurement of fractional shortening. In conclusion, these results provide the first documentation of alterations in S1P-mediated signaling during the in situ development of cardiomyopathy and suggest a possible therapeutic role for the pharmacological S1P receptor agonism in the post-MI heart.

Myocardial survival signaling in response to stem cell transplantation.

BACKGROUND Experimental human stem cell transplantation to the heart has begun, but the mechanisms underlying benefits seen in preclinical models, both at the site of cell injection and at more distant regions, remain uncertain. We hypothesize that these benefits can be best understood first at the level of key intracellular signaling cascades in the host myocardium, which can be responsible for functional and structural preservation of the heart. STUDY DESIGN Western blot and ELISA were used to assess key pathways that regulate cardiac myocyte survival and hypertrophy in both the infarct/borderzone and remote myocardium of C57/B6 mouse hearts subjected to coronary artery ligation, with subsequent injection of either vehicle or bone marrow-derived adult mesenchymal stem cells (MSC). RESULTS Improved left ventricular function with MSC transplantation was associated with a relative preservation of Akt phosphorylation (activation) and of phosphorylation of downstream mediators of cell survival and hypertrophy. There was no substantial difference in activation of mitogen-activated protein kinase p38, and activation of the antiapoptotic mitogen-activated protein kinase extracellular signal-regulated kinase was lower at 1 week after MSC treatment, but rose beyond controls by week 2. Similar changes were observed in both the infarct/borderzone and the remote myocardium. CONCLUSION Stem cell transplantation in the post-MI murine myocardium is associated with preservation of Akt signaling. Together with a possible later increase in extracellular signal-regulated kinase activation, this signaling change might be responsible for cardioprotection. Additional focused investigation might identify elements in transplantation regimens that optimize this mechanism of benefit, and that can increase the likelihood of human clinical success.

MEK1-induced physiological hypertrophy inhibits chronic post-myocardial infarction remodeling in mice.

Although activation of MEK‐ERK signaling is known to be cardioprotective during acute reperfusion injury, the effect of MEK activation on chronic changes in ventricular structure and function during the more complex process of remodeling after myocardial infarction (MI) with or without reperfusion remains uncertain. Four weeks after permanent coronary ligation, LV fractional shorting, preload recruitable stroke work, and end‐systolic elastance were all preserved in transgenic mice with CM‐specific upregulation of the MEK1‐ERK1/2 signaling pathway (MEK1 Tg) compared to wildtype (WT) controls (5.8% decline vs. 17.3%, P < 0.01; 603 ± 98 mmHg vs. 335 ± 98, P < 0.05; 6.14 ± 0.57 mmHg/µl vs. 3.92 ± 0.60, P < 0.05, respectively). Despite similar initial infarct sizes, post‐MI remodeling was significantly reduced in MEK1 Tg, demonstrated by reductions in chronic infarct size (28.5 ± 3.1% vs. 47.8 ± 3.2%), myocardial fibrosis (3.98 ± 0.74% vs. 9.27 ± 1.97%) and apoptosis (0.66 ± 0.11% vs. 1.60 ± 0.34%). Higher phosphorylation (i.e., activation) of pro‐survival transcription factor STAT3, higher expression of anti‐apoptotic protein Bcl2, and higher phosphorylation (i.e., inactivation) of pro‐apoptotic BAD were observed in the post‐MI remote myocardium of MEK1 Tg. MMP2 activity was higher in MEK1 Tg, while expression of TIMP3 and MMP9 activity were lower in transgenic mice. Beyond any immediate cardioprotective effect, therapeutic activation of MEK1‐ERK1/2 signaling during the chronic post‐MI period may preserve LV function by increasing the expression of pro‐survival factors and by suppressing factors, such as the balance between matrix modulating proteins, that promote pathological remodeling in the remote myocardium. J. Cell. Biochem. 114: 47–55, 2012.

Lung tumourigenesis in a conditional Cul4A transgenic mouse model

Cullin4A (Cul4A) is a scaffold protein that assembles cullin–RING ubiquitin ligase (E3) complexes and regulates many cellular events, including cell survival, development, growth and cell cycle control. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Here, we used a Cul4A transgenic mouse model to study the potential oncogenic role of Cul4A in lung tumour development. After Cul4A over‐expression was induced in the lungs for 32 weeks, atypical epithelial cells were observed. After 40 weeks, lung tumours were visible and were characterized as grade I or II adenocarcinomas. Immunohistochemistry (IHC) revealed decreased levels of Cul4A‐associated proteins p21CIP1 and tumour suppressor p19ARF in the lung tumours, suggesting that Cul4A regulated their expression in these tumours. Increased levels of p27KIP1 and p16INK4a were also detected in these tumours. Moreover, the protein level of DNA replication licensing factor CDT1 was decreased. Genomic instability in the lung tumours was further analysed by the results from pericentrin protein expression and array comparative genomic hybridization analysis. Furthermore, knocking down Cul4A expression in lung cancer H2170 cells increased their sensitivity to the chemotherapy drug cisplatin in vitro, suggesting that Cul4A over‐expression is associated with cisplatin resistance in the cancer cells. Our findings indicate that Cul4A is oncogenic in vivo, and this Cul4A mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A. Published by John Wiley & Sons, Ltd

Spacial and Temporal Patterns of Gene Expression After Cardiac MEK1 Gene Transfer Improve Post-Infarction Remodeling Without Inducing Global Hypertrophy.

Alteration of mitogen activated protein (MAP) kinase signaling in transgenic mice can ameliorate post‐myocardial infarction (MI) remodeling. However, pre‐existing changes in transgenic hearts and clinically unrealistic transgene expression likely affect the response to injury; it is unknown whether clinically relevant induction of transgene expression in an otherwise normal heart can yield similar benefits. Constitutively active MEK1 (aMEK1) or LacZ adeno‐associated virus 9 (AAV9) vectors were injected into the left ventricular (LV) chambers of mice either just before or after coronary ligation. Hearts were evaluated via Western blot, quantitative polymerase chain reaction, histology, and echocardiography. AAV9‐mediated aMEK1 delivery altered ERK1/2 expression/activation as in transgenic mice. Transgene expression was not immediately detectable but plateaued at 17 days, and therefore did not likely impact acute ischemia as it would in transgenics. With AAV9‐aMEK1 injection just prior to MI, robust expression in the infarct border zone during post‐MI remodeling increased border zone wall thickness and reduced infarct size versus controls at 4 weeks, but did not induce global hypertrophy. Significant improvements in local and global LV function were observed, as were trends toward a preservation of LV volume. Delivery after ligation significantly lowered transgene expression in the infarct border zone and did not yield structural or functional benefits. The primary benefits observed in transgenic mice, ameliorated remodeling, and reduced chronic infarct size, were achievable via clinically relevant gene transfer of aMEK1, supporting ongoing translational efforts. Important differences, however, were observed, and consideration must be given to the timing and distribution of transgene delivery and expression. J. Cell. Biochem. 118: 775–784, 2017.

Surgical ventricular reconstruction in mice: Elucidating potential targets for combined molecular/surgical intervention

OBJECTIVES We hypothesize that persistent alterations in molecular signaling may drive recurrent pathologic remodeling even after the reduction of mechanical stress achieved via surgical ventricular reconstruction. We developed a murine model of surgical ventricular reconstruction that would facilitate molecular analysis of the postreconstruction myocardium and allow future exploitation of genetic models. METHODS C57/B6 mice underwent coronary artery ligation. For surgical ventricular reconstruction at 4 weeks after myocardial infarction, a purse-string suture (7-0 polypropylene) achieved at least partial exclusion of the apical aneurysm. Serial echocardiography was correlated to measurements of apoptosis and to Western blot analysis of key signaling cascades. RESULTS An immediate 21.7% +/- 2.6% improvement in fractional shortening was seen in the remaining myocardium after surgical ventricular reconstruction. Reduction in left ventricular volume and improved function persisted at 1 week, but recurrent dilatation at 4 weeks (left ventricular end-diastolic volume of 63.5 +/- 2.5 vs 42.1 +/- 5.4 microL immediately after reconstruction; P < .05) was associated with a loss of functional improvement (fractional shortening 41.2% +/- 2% vs 46% +/- 0.9%; P < .01). At 1 week after surgical ventricular reconstruction, there was a transient reduction in myocardial apoptosis. A steady reduction in cardioprotective myocardial Akt activation, however, was not affected by ventricular reconstruction. CONCLUSION This murine model recapitulates both the immediate benefits of surgical ventricular reconstruction and the longer-term recurrence of dilated cardiomyopathy seen previously in some animal models and human studies. Early analysis has begun to implicate persistent signaling changes in the postinfarction myocardium that may be responsible for recurrent dilatation after surgical ventricular reconstruction and that may become targets for combined surgical and molecular interventions.

Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐rasG12C mutation) and PC9‐BrM3 (EGFRΔexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐rasG12C mutation) and PC9 (EGFRΔexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.