The Daily Sign-Out

ETS2 mediates tumor suppressor function and MET inhibition in lung adenocarcinoma

There was a very intriguing paper published online by Clinical Cancer Research on May 9 last week that should be of interest for those involved in lung cancer research.  The authors are Mohamed Kabbout, Melinda Garcia, Junya Fujimoto, et al. from M.D. Ancerson Cancer Center in Houston and the title of the article is "ETS2 mediated tumor suppressive function and MET oncogene inhibition in
human non-small cell lung cancer." 

In brief, the authors find that there is significant downregulation of the ETS2 transcription factor in lung adenocarcinomas compared to normal lung tissues.  ETS2 expression is linked to various canonical cancer-associated pathways in lung cancer cells, in particular the HGF pathway.  Moreover, knockdown of ETS2 is associated with up-regulation of the ZEB1, SNAI2, RAB3B, SERPINB5 and SPDEF genes
with concomitant with elevated cell migration and invasion.  Last, low ETS2 protein is predictive of shorter time-to-recurrence in NSCLC and lung adenocarcinoma.  These data suggest that ETS2 functions as a tumor-suppressor in lung cancer cells by, at least in part, regulation of the migration- and invasion-promoting transcriptome.

More details. . .

Gene expression profiling showed significant decreased expression of ETS2 transcription factor transcripts in lung adenocarcinoma (ADC) compared to normal controls and quantitative RT-PCR confirmed decreased ETS2 expression.  Subset analysis should significantly decreased ETS2 expression in those ADCs from smokers compared to those from never-smokers.

They analyzed ETS2 expression by IHC in a microarray set of 201 NSCLC (135 ADC, 66 squamous cell carcinoma-SQC) tumors.  Low ETS2 expression was associated with shorter time-to-recurrence in stage I and all stages of patients.  Multivariate Cox proportional hazards analysis showed that, after adjusting for stage, low ETS2 expression was an independent predictor of shorter time-to-recurrence in NSCLC and ADC.

H441 human lung cancer cell lines transfected with small interfering RNA (siRNA) specific to ETS2 showed significantly reduced ETS2 transcript compared to controls.  ETS2 knockdown significantly increased cell growth, migration, and invasion.  Conversely, ETS2 overexpression in H1299 lung cancer cells showed significantly decreased cell migration and invasion.  Of interest, the magnitude of these changes following knockdown or overexpression of ETS2 was greater in measures of migration and invasion compared to cell growth--suggesting a tumor suppressor function for ETS2 at least in vitro.

Gene expression profiling of H441 lung cancer cell lines transfected with scrambled siRNA (control) versus ETS2-specific siRNA showed significant differential expression in 1,816 transcripts.  Functional analysis by Ingenuity Pathways Analysis (IPA) showed ETS2 knockdown modulated key oncogenic pathways, including HGF, integrin, tissue factor, semaphorin, and MAPK signaling.  Notably, HGF was the top modulated canonical pathway identified following knowdown, and further, an HGF-mediated gene-interaction network was among the among the top significant gene networks following topological arrangement of the genes found to be differentially expressed by IPA.

Western blot analysis and ELISA both showed increased phosphorylation MET following ETS2 knockdown.  Importantly, co-transfection of H441 lung cancer cells with MET-specific siRNA abrogated cell migration, invasion, and, to a lesser effect, cell growth mediated by knockdown of ETS2 expression alone.  Furthermore, co-transfection of cells with MET-specific siRNA significantly attenuated the induction of SNAI2, RAB3B, SPDEF and SERPINB5 mediated by knockdown of ETS2 alone as well as reduced expression of ZEB1. These data demonstrate that ETS2 suppresses key features of the lung cell malignant phenotype, at least in part, through inhibition of the MET oncogene. 

The authors also find that ETS2 suppresses EGF-mediated signaling in lung cancer cells.  HGF treatment increased ETS2 protein expression and phosphorylation of MET and ERK1/2 MAPK in H441 cells.  Conversely, expression of ETS2, phospho-MET, and phospho-ERK1/2 was attenuated by co-treatment of H441 cells with the MET TKI PHA-665752.  HGF treatment also significantly increased the expression of SNAI1, RAB3B, SPDEF, ZEB1, and SERPINB5 genes.  Moreover, the expression of these genes was significantly higher in HGF-treated cells transfected with ETS2-specific siRNA compared to similarly treated control cells.  Conversely, overexpression of ETS2 in H1299 cells decreased HGF-induced phosphorylated MET levels and decreased HGF-induced cell migration in the wound healing assay. 

Finally, NSCLC (p=0.01) or ADC (p=0.02) patients with relatively low ETS2 protein expression and higher phospho-MET membrane immunoreactivity showed shortest tiime-to-recurrence compared to other patient groups, specifically those with higher ETS2 and higher phospho-MET
expression.

Epithelial-Mesenchymal Transition and Lung Cancer: Mechanism for Resistance to EGFR-TKIs?

Despite all of the encouraging clinical results with molecularly targeted therapies against non-small cell lung cancer, there are still important and significant questions that continue to vex.  The discovery of activating mutations in EGFR radically changed the treatment options for these patients, and, indeed, treatment of advanced stage patients with EGFR-mutated tumors with erlotinib (in the US) is now front-line therapy.  However, even patients who initially show dramatic responses invariably relapse due to the development of drug resistance.  Although such resistance can be explained in about 50% of these patients, what are the mechanisms for the other 50%?  Conversely, why do some, albeit rare, patients who are wild-type for EGFR respond to EGFR-TKIs?  Also, why don't conventional chemotherapy and molecularly targeted therapy produce a synergistic effect?  In fact, patients treated with combined therapy seem to actually do worse than if they get just one or the other--why?

Numerous research groups are trying to identify more effective biomarkers to identify predictive markers for response or resistance to EGFR-TKIs in order to optimize therapy for individual patients.  Our group at Rush University Medical Center has been focused on the role of epithelial-mesenchymal transition as a prognostic marker for early-stage NSCLC but also as a predictive marker for response to EGFR-TKIs.  So I read with great interest the article by Byers et al. recently published online in Clinical Cancer Research on December 20, 2012 (abstract here).

Some important notes:

 

• The EMT first principal component correlated better with E-cadherin protein level than did even the best CDH1 mRNA probe set.
• Mesenchymal cell lines were highly resistant to erlotinib as compared to epithelial cell lines, which agrees with previous work.  It is interesting, however, that while cell lines with EGFR-activating mutations were the most sensitive to erlotinib, in a subset of 40 cell lines WT for both EGFR and KRAS, the correlation between EMT signature and erlotinib resistance was still observed with significantly greater resistance in mesenchymal-like cell lines.  An important corollary is that the EMT signature was a better predictor of erlotinib response than mRNA probe sets for either CDH1 (E-cadherin) or VIM genes individually--a finding that agrees with the work we've done in our lab.
• EMT does not appear to be a general marker of resistance.  Indeed, mesenchymal cells were not more resistant to other targeted agents, such as sorafenib or to commonly used cytotoxic chemotherapies, including pemetrexed, docetaxel, paclitaxel, and platinum-doublets. Instead, a trend toward greater relative sensitivity was seen in mesenchymal cells as compared with epithelial for cisplatin. 
• An EMT signature may be a predictive marker in EGFR wild-type/KRAS wild-type patients with advanced stage NSCLC.  The authors analyzed the association between EMT signature and clinical outcome in patients enrolled in the BATTLE-1 trial but limited their analysis to patients whose tumors were WT for EGFR and KRAS.  Although the N is only 20, patients with disease control at 8 weeks (the primary study endpoint) showed a more epithelial-like signature as compared with those without disease control, although the difference is of borderline significance (P=0.05, by t test).  However, among the full group of 101 of 139 clinically evaluable patients (all treatment arms), expression of EMT signature genes was not prognostic of 8-week disease control or progression-free survival (PFS) in the overall group (all treatment arms).

This is a dense article but well-worth reading through in detail if you are interested in either EMT in NSCLC or mechanisms of EGFR-TKI drug resistance.  I offer a few further comments.

First, note that this is a study of NSCLC cell lines, so the findings have to be taken as hypothesis-generating for confirmation in clinical tumor specimens.  The limitations of studying cell lines ought to be well-known to you all.  But one point that has to be appreciated is that the phenomenon of EMT in vivo is context-dependent and critically depends on interaction with the tumor microenvironment--and this crucial fact is not and cannot be studied in this type of model.  Second, although it seems obvious to state this, histological subtype matters greatly with regard to EGFR mutations, EMT, and response to erlotinib.  Why do so many recently published studies still lump together non-small-cell lung cancer?  We recently focused exclusively on a group of patients with resected lung adenocarcinomas who received erlotinib upon disease progression in a study before the advent of EGFR mutation testing.  We identified two separate "epithelial" (high E-cadherin/low vimentin) and "mesenchymal" (low E-cad/high VIM) as well as intermediate/transitional tumors and found a trend toward worse DFS that did not quite reach statistical significance.  Unfortunately, our study was small (N=45) and I think that much larger numbers will be needed to show any importance difference.  In this study, out of 35 cell lines with adenocarcinoma histology, 29 (83%) had epithelial signatures, while 8 (23%) had mesenchymal signatures. Of 4 cell lines with squamous histology, there was an even distribution between epithelial and mesenchymal subsets.  Thus, the adenocarcinomas more commonly expressed an epithelial signature (P<0.0016, Chi-square test).  Finally, I have observed in our studies that EMT in vivo is a heterogenous process throughout a tumor.  Indeed, even if you focus on the invasive edge of a tumor, where one expects EMT to be occurring (as we did in a recently published abstract at AACR 2012), there is frequently significant variation in the expression and distribution of E-cadherin and vimentin.  What is the significance of this variation?  Can it be quantified and does the amount of variation in EMT have any prognostic or predictive significance?  Don't know--image analysis and quantitative immunohistochemical studies could possibly answer these questions. 

CAP Public Comment Period for New Biomarker Reporting Checklists

Take a look at the new cancer biomarker reporting checklists proposed by the College of American Pathologists for breast, colon and lung cancers.  You don't have to be a CAP member to provide comments (but I encourage you to join CAP anyway!).

In an effort to improve consistency and completeness in reporting results of cancer biomarker testing, the College of American Pathologists (CAP) has produced new cancer biomarker reporting templates for Breast, Lung, and Colon. These templates were developed primarily to respond to pathologist user feedback about timing of reporting and structural consistency of ancillary studies data elements in the CAP Cancer Protocols. The development of these templates has been a collaborative effort between the CAP, the Association for Molecular Pathology (AMP), the American Society of Clinical Oncology (ASCO), Centers for Disease Control and Prevention (CDC), the American Joint Committee on Cancer (AJCC), and many other participating organizations.

The cancer biomarker reporting templates are intended to encompass all important data elements for routinely assessed tumor markers for each site, as well as some optional markers. Once these templates are approved by the CAP for general use, they will replace the Ancillary Studies sections of their corresponding CAP cancer protocols.

Prior to the release of these new biomarker reporting templates, the CAP will host a public comment period. You can review the revisions and submit feedback online. The public comment period will run from December 3–31, 2012. Please note that submitted comments are not instantly available for review and will be posted on a weekly basis.

The Cancer Biomarker Reporting Workgroup would appreciate your feedback on these new biomarker reporting templates. Thank you in advance for taking the time to review these and submit your responses.

Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer

Thought this was an intriguing abstract. Signaling crosstalk between EGFR and integrins was a hot topic at AACC meeting earlier this year.




From Evernote:




Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer
Clipped from: http://clincancerres.aacrjournals.org/content/early/2012/10/19/1078-0432.CCR-11-2162.abstract?papetoc



Impact of the Integrin Signaling Adaptor Protein NEDD9 on Prognosis and Metastatic Behavior of Human Lung Cancer

Shunsuke Kondo1,Satoshi Iwata2, Taketo Yamada4, Yusuke Inoue7, Hiromi Ichihara3, Yoshiko Kichikawa2, Tomoki Katayose2, Akiko Souta-Kuribara2, Hiroto Yamazaki2, Osamu Hosono2, Hiroshi Kawasaki2, Hirotoshi Tanaka2, Yuichiro Hayashi4, Michiie Sakamoto4, Kazunori Kamiya5, Nam H. Dang8, and Chikao Morimoto6+
Authors' Affiliations: 1Hepatobiliary and Pancreatic Oncology Division, National Cancer Center Hospital; 2Department of Rheumatology and Allergy, Research Hospital, 3Division of Molecular Pathology, The Institute of Medical Science, The University of Tokyo; 4Department of Pathology, School of Medicine, 5Department of Pathology and Department of Surgery, Division of General Thoracic Surgery, School of Medicine, Keio University;
6Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University,
Tokyo; 7Department of Diagnostic Radiology, Kitasato University Hospital, Kanagawa, Japan; and 8Division of Hematology and Oncology, University of Florida Shands Cancer Center, Gainesville, Florida


Corresponding Author:
Chikao Morimoto, Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine,
Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-3868-2310; Fax: 81-3-3868-2310; E-mail: [email protected]


Abstract

Purpose: In a substantial population of non–small cell lung cancer (NSCLC), expression and activation of EGF receptor (EGFR) have been reported and is regarded as a novel molecular target. A growing body of evidence has shown the signaling crosstalk between EGFR and integrins in cellular migration and invasion. NEDD9 is an integrin signaling adaptor protein composed of multiple domains serving as substrate for a variety of tyrosine kinases. In the present study, we aimed at elucidating a role of NEDD9 in the signaling crosstalk between EGFR and integrins.


Experimental Design: Using NSCLC cell lines, we conducted immunoblotting and cellular migration/invasion assay in vitro. Next, we analyzed metastasis assays in vivo by the use of xenograft transplantation model. Finally, we retrospectively evaluated clinical samples and records of patients
with NSCLCs.


Results: We showed that tyrosine phosphorylation of NEDD9 was reduced by the inhibition of EGFR in NSCLC cell lines. Overexpression of constitutively active EGFR caused tyrosine phosphorylation of NEDD9 in the absence of integrin stimulation. By gene transfer and gene knockdown, we showed that NEDD9 plays a pivotal role in cell migration and invasion of those cells in vitro. Furthermore, overexpression of NEDD9 promoted lung metastasis of an NSCLC cell line in NOD/Shi-scid, IL-2Rγnull mice (NOG) mice. Finally, univariate and multivariate Cox model analysis of NSCLC clinical specimens revealed a strong correlation
between NEDD9 expression and recurrence-free survival as well as overall survival.


Conclusion: Our data thus suggest that NEDD9 is a promising biomarker for the prognosis of NSCLCs and its expression can promote NSCLC
metastasis. Clin Cancer Res; 1–13. ©2012 AACR.



Footnotes

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).



Received August 23, 2011.
Revision received August 20, 2012.
Accepted August 28, 2012.

©2012 American Association for Cancer Research.