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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000034
Original Articles

Erk/MAP Kinase Signaling Pathway and Neuroendocrine Differentiation of Non–Small-Cell Lung Cancer

Chen, Yuhchyau MD, PhD*; Nowak, Irena PhD*; Huang, Jiaoti MD, PhD; Keng, Peter C. PhD*; Sun, Hongliang PhD*; Xu, Haodong MD, PhD; Wei, Gang MD, PhD*; Lee, Soo Ok PhD*

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Author Information

*Department of Radiation Oncology, University of Rochester Medical Center, James P. Wilmot Cancer Center, Rochester, New York; Department of Pathology & Laboratory Medicine, Institute of Molecular Medicine, University of California, Los Angeles, California; and Department of Pathology & Laboratory Medicine, University of Rochester Medical Center. Rochester, New York.

Disclosure: The authors declare no conflict of interest.

Address for correspondence: Yuhchyau Chen, MD, PhD, University of Rochester, 601 Elmwood Avenue Box 647, Rochester, NY 14642. E-mail: Yuhchyau_Chen@urmc.rochester.edu

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Abstract

Introduction: Almost all small-cell lung cancers (SCLC) and carcinoid tumors express neuroendocrine differentiation (NED), and 10% to 20% of non–small-cell lung cancers (NSCLC) are associated with NED. Although distinct clinical features and histology of SCLC and carcinoid tumors are well recognized, the clinical significance and the molecular basis of NED in NSCLC remain unclear.

Methods: To explore the potential molecular pathway involved in NED of NSCLC and its clinical relevance, we conducted investigations using an NSCLC cell line (NCI-H157) as a NED induction model, and explored the potential intracellular signal transduction pathways involved in NED of NSCLC. We confirmed our findings using activators versus inhibitors to these signal transduction pathways in vitro. We also performed immunohistochemical stains of phospho-Erk1/2 of lung cancer specimens known to have NED and explored its clinical relevance.

Results: We discovered that NED of NSCLC was associated with the activation of Erk1/2-mitogen-activated protein kinases (MAPK) signal transduction pathway, and the inhibition of the Akt signal transduction pathway. Using specific activator (Pb2+) and inhibitors (siRNA-Erk1/2 and U0126) to the Erk1/2-MAP-kinase pathway, as well as the inhibitor (LY294002) to the Akt pathway, we found that Erk1/2-MAP-kinase activation was essential for NED of NCI-H157 cells. Staining of Erk1/2-MAP-kinase pathway revealed a high rate of positivity in NSCLC tumors with NED when compared with other neuroendocrine lung tumors.

Conclusions: To our knowledge, our findings are the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in the association with NED. Further investigation of the Erk/MAPK signal transduction pathway of NSCLC may yield discoveries in identifying specific molecular targets for the treatment of NSCLC with NED.

Lung cancer is the leading cause of cancer death among all cancer types. There are two general categories of lung cancer: small-cell lung cancer (SCLC) and non–small-cell lung cancer (NSCLC), which are differentiated by histologic appearance, clinical behavior, and cancer therapy responses. Although SCLC constitutes 15% to 20% of lung cancer diagnoses, NSCLC is much more common and is composed of heterogenous histologic subtypes including adenocarcinoma, squamous cell carcinoma, and large-cell carcinoma.1

Neuroendocrine differentiation (NED) has been observed in approximately 25% to 33% of all lung tumors.2,3 Essentially, all SCLC and carcinoid tumors show distinct histological structure and stain positive to neuroendocrine markers such as neuroamines, neuropeptides, dense core secretory granules, chromogranin A, neuroendocrine-specific protein, and neuron-specific enolase (NSE).3–9 It was later recognized that NED was not limited to SCLC and carcinoid, and it could involve NSCLC such as large-cell neuroendocrine carcinoma (LCNEC).10 Subsequently, it was found that about 10% to 20% of NSCLC, including adenocarcinomas and squamous cell carcinomas, also exhibit some neuroendocrine properties despite being considered nonneuroendocrine types.2,11 It has been postulated that NED of NSCLC represents an intermediary transition between SCLC and NSCLC,12 or that NED may be a predictor of the response to chemotherapy or radiotherapy,13,14 as well as a predictor of shorter survival in patients with stage I adenocarcinoma of the lung.3,15 Irrespective of these observations, the clinical significance of NED of NSCLC remains unclear.

For SCLC, the mitogen-activated protein kinases (MAPKs) pathway via Raf and activated protein kinase C has been implicated in the regulation of growth and differentiation through neuropeptides.16–18 Although much is known of the NED of SCLC, the molecular basis of NED in NSCLC is not well understood. Erk/MAPKs are widely expressed serine-threonine kinases comprising the three major subfamilies: extracellular-signal regulated kinases Erk 1 and 2 (Erk1/2), c-Jun N-terminal kinases (JNKs), and p38 kinase. The Erk1/2-MAPK (p44/42) signaling pathway can be activated in response to many factors like mitogens, growth factors, and cytokines.19 These are involved in many cellular events, although their functions in regulating specific responses are quite different depending on the cell types.20,21 In vitro investigation of prostate cancer has shown that the activation of the Erk/MAP kinase pathway stimulates NED of LNCaP cells.22 Constitutive activation of the MAPK pathway has been shown to contribute to malignant transformation of mammalian cells, and has been associated with aggressive neoplastic phenotype and cancer resistance to chemotherapy.23–25

We describe in this report, using an NSCLC cell line in vitro NED induction model, that the Erk/MAPK signaling pathway may be involved in NED of NSCLC. We also explored whether another major intracellular signal pathway PI3-kinase/Akt pathway was involved, as both Erk1/2-MAPK pathway and PI3-kinases/Akt pathways were major intracellular signaling modules. To our knowledge, the investigation of molecular signaling pathways involving Erk/MAPK for NED of NSCLC has not been reported in the published literature. Because there has been interest in developing anticancer drugs that target the MAPK pathway in the past decade26–28 we think the investigation of the intracellular signal transduction pathway involved with NED of NSCLC cells may reveal potential molecular targets for the treatment of NSCLC with NED.

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MATERIALS AND METHODS

Materials

The human NSCLC squamous cell line NCI-H157 was obtained from Dr. Oh (Virginia Commonwealth University, Richmond, VA). RPMI1640, fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA); 8-bromoadenosine 3′,5′ cyclic monophosphate (cAMP), 3-isobutyl-1-methylxanthine (IBMX) and Bradford Reagent were from Sigma (St Louis, MO); monoclonal anti-NSE antibody was from DAKO (Carpinteria, CA); monoclonal glyceraldehyde-3-dehydrogenase, polyclonal Akt1,2,3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); siRNA-Erk1/2, monoclonal antiphospho Akt (S473), anti-Erk 1/2, and antiphospho-Erk 1/2(T202/Y204) were from Cell Signaling Technology (Danvers, MA); and radio-immunoprecipitation assay (RIPA) lysis buffer was from Upstate Cell Signaling Solution (Lake Placid, NY). U0126 (used at a final concentration of 10 µM) was from Calbiochem (San Diego, CA); LY294002 (used at final concentration of 20 µM) was from Cayman Chemical (Ann Arbor, MI); and Interfere-in Transfection Reagent was from Polyplus-Transfection (Illkirch, France).

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Cell Culture and the Induction of NED

The NSCLC NCI-H157 cell line was cultured in RPMI 1640 medium supplemented with 20 mM glutamine, 25mM HEPES, and 10 % FBS. Cells were first serum-starved by overnight incubation with serum-free medium (SFM) containing 1 % bovine serum albumin. For the induction of NED, overnight serum-starved cells were treated with SFM containing 0.5 mM IBMX/0.5mM cAMP for 72 hours as described previously.29 For the cells without IBMX/cAMP treatment, the overnight serum-starved cells were treated with fresh SFM for the next 72 hours without the addition of IBMX/cAMP.

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Western Blotting

Cells were washed twice with cold Dulbecco's phosphate buffered saline (DPBS) and lysed in RIPA buffer, scraped from the tissue culture plate, sheared through a 20-gauge needle, then centrifuged at 14,000 rpm for 10 minutes. The cellular protein concentration in the cell lysates was determined using the Bradford assay (Sigma, St. Louis, MO). Proteins were then separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to the nitrocellulose membrane using Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA). Membranes were blocked with Tris-Buffered Saline/0.5% Tween 20 containing 5% nonfat dry milk, incubated with the desired primary antibody diluted with Tris-Buffered Saline/0.5% Tween 20 containing 3% nonfat dry milk. After incubation with the appropriate secondary antibody, proteins were detected with Immun-Star Western C kit (Bio-Rad, Hercules, CA). The ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA) was used to analyze the densitometry of Western blots.

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siRNA Transfection

siRNA-Erk1/2 transfections were performed at 20 nM using the Interfere-in transfection reagent (Polyplus Transfections, Illkirch, France). The cells (1 × 106) were plated in 10-cm plates 24 hours before experiment to reach density 30% to 50% at the day of the experiment and then transfected with siRNA-Erk1/2 according to the manufacturer protocol. An Opti-MEM reduced serum medium (Invitrogen) was used during transfection.

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Immunohistochemical Staining

Immunohistochemical (IHC) staining of lung cancer specimens was performed under the purview of a protocol approved by the Internal Review Board (PI: Haodong Xu). For IHC staining, sections (5 µm thick) were cut from formalin-fixed and paraffin-embedded tissue specimens mounted on glass microscopic slides and air-dried overnight and then incubated at 60°C for 1 hour. After conventional deparaffinization, the indirect IHC staining was performed. In brief, slides were subjected to an antigen retrieval procedure in 10 mM sodium citrate buffer, pH 6.0 at 99°C for 20 minutes, and then cooled to 60°C and then endogenous peroxidase was quenched in 3% hydrogen peroxide for 10 minutes. Next, the slides were incubated with the rabbit monoclonal antibody phospho-Erk1/2 (Thr202/Tyr204; Cell Signaling Technology, Danvers MA) diluted 1:400 for 30 minutes, followed by Flex-HRP Detection kit according to the manufacturer instructions (Dako, Carpinteria, CA) and then visualized with liquid 3,3′diaminobenzidine (DAKO), counterstained with hematoxylin and rinsed with PBS. The reading of the phospho-Erk1/2 stain was conducted by a pathologist on coded sample specimens.

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Statistical Analysis

Statistical analysis was performed using the two-tail t test.

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RESULTS

NED: Erk1/2 and Akt Phosphorylation

NED of NCI-H157 cells was induced in SFM containing NED induction compounds 0.5mM IBMX/0.5mM cAMP. The results presented as part of Figures 1AC were protein expression by Western blot analysis. Figure 1A shows a marked increase of NSE expression after 72 hours of incubation in SFM containing 0.5 mM IBMX/0.5mM cAMP. The incubation of cells in SFM without the differentiating agents IBMX/cAMP seemed to also induce some NSE expression, but to a much lesser extent and did not reach a statistically significant level (p=0.24). Figure 1B shows that the NED of NCI-H157 cells were accompanied by an increased phosphorylation of Erk1/2. In contrast, Akt was constitutively active in control cells (serum-starved overnight), and to a lesser extent in cells incubated in SFM without the differentiating agents IBMX/cAMP (Fig. 1C). Cells with induced NED by IBMX/cAMP have much lower level of phosphorylated Akt, suggesting that Akt phosphorylation was inhibited in cells with NED (Fig. 1C).

Figure 1
Figure 1
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Effect of U0126 on NSE Expression

U0126 is a highly selective inhibitor of MEK (MAP kinase) kinase (a kinase upstream of Erk1/2). On the basis of findings from Figure 1 that NED was associated with Erk1/2 activation, we tested whether activation of Erk1/2 kinase was essential for NED of NCI-H157 cells. We induced NED of NCI-H157 cells with 0.5mM IBMX/0.5mM cAMP in the presence of 10 µM of U0126 to assess the effect of inhibition of Erk1/2 by U0126. Figure 2A demonstrates that U0126 did inhibit Erk1/2 phosphorylation, whereas Figure 2B shows that U0126 also inhibited NSE expression in NED cells. Our finding using U0126 further supported that Erk1/2 signaling pathway was essential in the in vitro induction of NED of NCI-H157 cells.

Figure 2
Figure 2
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Effect of Inhibition of Akt Phosphorylation on NSE Expression

In Figure 1C, we showed that NED of NCI-H157 cells was accompanied by the inhibition of Akt phosphorylation. It remained unclear whether the inhibition of Akt phosphorylation might lead to the NED of NCI-H157 cells. To test whether there was any causal relationship, we examined the effect of Akt phosphorylation inhibition by LY294002 (20 µM) on NCI-H157 cells. Figure 3 shows that inhibition of Akt activation of NCI-H157 cells did not increase NSE expression in these cells. Such an observation demonstrates that Akt pathway was not essential for the NED of NCI-H157 cells.

Figure 3
Figure 3
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Effect of Silencing Erk1/2 Gene on NSEExpression in Neuroendocrine-Differentiated NSCLC Cells

To provide more direct evidence that the activation of the Erk1/2-MAPK pathway was essential for NED of NCI-H157 cells, we applied the siRNA approach to silence the expression of the Erk1/2 protein. We sought to examine whether silencing Erk 1/2 gene expression might have any effect on NSE expression of these cells. Figure 4A shows that application of siRNA to silence the Erk1/2 gene significantly reduced the expression of Erk1/2 in NCI-H157 cells. Figure 4B shows that siRNA treatment to silence the Erk1/2 gene expression significantly decreased NSE expression in NCI-H157 cells.

Figure 4
Figure 4
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Pb2+ Effect on Erk1/2 Activation and NSE Expression

It has previously been shown that Pb2+ ions can cause prolonged activation of Erk1/2 in lung cancer cell line CL3.30 To provide further evidence that Erk1/2-MAPK was involved in NED of NCI-H157 cells, we treated these cells with Pb2+ (30 µM) and assessed the effect on Erk1/2 activation. We found that Pb2+ treatment led to a period of Erk1/2 activation, with maximum phosphorylation of Erk1/2 observed at 30 minutes after treatment. After the peak level of activation, the level of Erk1/2 phosphorylation declined over time and reached the baseline level by 24 hours post Pb2+ treatment (Fig. 5A). Pb2+ treatment also led to an increased expression of NSE, with the maximum effect at 6 hours after Pb2+ treatment (Fig. 5B). Such data lent further support of the involvement of Erk1/2 activation in the NED of NCI-H157 NSCLC cells.

Figure 5
Figure 5
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Phosphorylated Erk1/2 Expression in Lung Tumor Specimens

To determine whether the pathway of Erk1/2 pathway activation discovered in the lung cancer cell is relevant in NED of human NSCLC, we examined 39 lung tumor samples known to have NED, which included typical and atypical carcinoid, SCLC, and NSCLC. We conducted IHC staining for phosphorylated Erk1/2 expression of these tumors. Erk1/2 positivity was defined by 95% or more tumor cells staining positive with phospho-Erk1/2 We found out that the positive rate of IHC was 0 of 10 (0%) for typical carcinoid; 2 of 10 (20%) for atypical carcinoid; 4 of 9 (44.4%) for small-cell lung carcinoma; and was 7 of 10 (70%) for LCNEC. Examples of the hematoxylin and eosin stain and IHC stain for different histologies are shown in Figure 6. The Erk1/2 activation was statistically different (**p<0.01 by two-tailed Fisher’s exact test) comparing all carcinoid tumors (2 of 20, typical plus atypical) with high-grade neuroendocrine carcinoma (11 of 19, SCLC plus NSCLC). The differences between individual histology types did not reach significance, likely because of small sample sizes.

Figure 6
Figure 6
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DISCUSSION

NED is observed in essentially all SCLC of the lung, which exhibits distinct histopathologic appearances and clinical behavior. SCLC is very sensitive to both chemotherapy and radiation treatment whereas NSCLC is not. Among all NSCLC, NED of NSCLC has been observed in approximately 10 to 20% of conventional adenocarcinoma, squamous cell carcinoma, or large-cell carcinoma, but the clinical significance and optimal treatment of NSCLC with features of NED remain unclear. Further, the pathogenesis of NED in NSCLC is not clearly understood. Different studies have attempted to address the prognostic value of NED in NSCLC. Some reported that NED in NSCLC identified a subset of tumors with poorer prognosis and reduced life expectancy in patients with stage I pulmonary adenocarcinomas.3 Others reported that NED might be of prognostic significance in patients with advanced-stage NSCLC treated with chemotherapy.14 Yet other studies indicated that there was no correlation between NED of NSCLC and disease-specific and overall survival.31–34

Erk/MAPK pathway through Raf-1 and protein kinase C activation has been implicated in the regulation of growth by neuropeptides regarding the neuroendocrine nature of SCLC,16,17 and may be responsible for the development of the great majority of neuroendocrine foci in lung cancer cells. In this report, we induced NED of the NSCLC cell line NCI-H157 and investigated the intracellular signal transduction pathways associated with NED. We would like to point out that the NCI-H157 cell line served only as an investigational model for the in vitro induction of NED, which allowed us to investigate potential molecular pathways involved with NED. This cell line has previously been used by other investigators for NED induction in vitro as well.29 We found that NED of NCI-H157 was associated with the activation of Erk1/2-MAPK pathway and the inhibition of the Akt pathway. Our data showed that the activation of Erk/MAPK pathway was essential to NED of the cell line NCI-H157 (Fig. 1) in that we consistently observed reduction of NSE expression (NED marker) after treatment of differentiating NCI-H157 cells with MEK inhibitor U0126 (Fig. 2), and with specific siRNA approach where expression of Erk1/2 protein was suppressed. The latter was correlated with the inhibition of NSE expression (Fig. 4). When cells were treated with LY294002, an inhibitor to Akt signal transduction pathway, we did not observe changes of the expression of NSE. Taken together, our data support that in this culture model, the activation of Erk1/2-MAPK signal transduction pathway was essential for NED of NCI-H157 cells, whereas the inhibition of the Akt pathway was not.

The ErK1/2-MAPK pathway can be activated in many cell types by a broad spectrum of extracellular stimuli causing different physiological responses, most often cell division proliferation, and differentiation.35,36 It has been shown that aberrant expression and/or mutations of the Erk/MAPK signal transduction pathway were associated with human cancer.19,21,37 Sustained activation of Erk1/2-MAPK via Ras-Raf-MEK1/2 has been reported in several types of cancer including kidney, colon, lung, prostate,37 and esophagogastric malignancies.38 Other studies20,38 have provided evidence that Erk/MAPK activation in cancer could be MEK independent, or that at least PI3K or conventional protein C kinase isoforms could contribute to MEK-independent Erk activation.

In our observation, the Erk1/2 activation was correlated well with human lung cancers that undergo NED. The choice of LCNEC was to take a clearly defined histologic type of NSCLC with NED to compare with lung cancer of other distinct histology with NED as well, that is, small cell and carcinoid. Findings from this article serve as the basis for us to extend the investigation to include specimens of NSCLC with unexpected NED irrespective of histologic subtype (such as adenocarcinoma and squamous cell carcinoma) for the ERk1/2 activation. The high rate (70%) of positivity of phosphorylated Erk1/2 stain seen in LCNEC, a type of NSCLC, and the much lower rates of positivity in other lung tumors known for NED (0% for typical carcinoid, 20% for atypical carcinoid and 40% for SCLC) is interesting. It proves that the Erk/MAPK signal transduction pathway is likely one of the mechanisms for the NED of NSCLC. SCLC positivity is noted at 44.4%, suggesting that the NED of some SCLC may share similar pathway of NED with NSCLC. We acknowledge that the sample size of 39 was small; additional investigations using a larger sample size will be more informative.

To our knowledge, our finding is the first to describe the potential involvement of Erk/MAPK signal transduction pathway of NSCLC in association with NED. Among the MAP kinase pathways, the inhibition of Erk/MAPK pathway has made some progress in drug development.39,40 For example, sorafenib was shown to block the Raf/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5.41 Future research in this direction may help to discover molecular targeted therapy for NSCLC with NED.

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ACKNOWLEDGMENTS

We thank Ms. Laura Finger for editorial assistance. This study was supported in part by Meaghan’s Hope.

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Non–small-cell lung cancer; Neuroendocrine differentiation; Cell signaling; Erk/mitogen-activated protein kinases

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